Antisense oligonucleotides and related methods for regulating cell death

ABSTRACT

This invention provides a first nucleic acid which specifically hybridizes to a nucleic acid encoding an inhibitor-of-apoptosis protein. This invention also provides related compositions and methods for inducing cell death and treating cancer using same. This invention further provides a second nucleic acid which specifically hybridizes to a nucleic acid encoding a protein, other than caspase-2, that induces cell death. Finally, this invention provides related compositions and methods for inhibiting cell death, inhibiting neuronal cell death in particular, and treating a neurodegenerative disorder and a heart disorder using the second nucleic acid.

[0001] This application is a continuation-in-part and claims priority of U.S. Ser. No. 09/898,158, filed Jul. 3, 2001, the contents of which are hereby incorporated by reference into this application.

[0002] The invention disclosed herein was made with government support under Grant Nos. N535933 and N515076 from the National Institutes of Health. Accordingly, the United States Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] Throughout this application various references are cited by author and date. Full bibliographic citations for these references may be found at the end of the Experimental Details section. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

[0004] During development, neurons that fail to find appropriate targets and sources of target-derived neurotrophic factors undergo apoptotic cell death (Pettmann and Henderson 1998). The elimination of improperly connected neurons constitutes a critical step in the formation of specific connections in the nervous system.

[0005] Caspases are an evolutionarily conserved family of proteins with at least 14 mammalian members (Thornberry and Lazebnik 1998). Data from caspase-null and Apafl-null mice support a role for the caspase-9 pathway in mediating death occurring early in the development of the nervous system (Kuida et al. 1996; Hakem et al. 1998; Kuida et al. 1998; Yoshida et al. 1998), when mainly neuroblasts are being removed. It is not clear which caspases are necessary for the removal of neurons which occurs later in development. It has been reported that the caspase-dependency of a pathway can be altered in caspase-null mice (Zheng et al. 2000) but no mechanism was provided for the observations described. Studies of the caspases required to execute trophic factor deprivation (TFD)-induced death of NGF-dependent neurons have given apparently conflicting results. In the case of cultured rat and mouse sympathetic neurons, as well as of neuronal PC12 cells, there is evidence that caspase-2 is necessary for TFD-induced death. Both acute downregulation of caspase-2 expression with an antisense oligonucleotide (Troy et al. 1997) and chronic downregulation of caspase-2 in PC12 cells by stable transfection with antisense caspase-2 (Haviv et al. 1998) protect these cells from NGF deprivation. In contrast, sympathetic neurons cultured from caspase-2-null mice retain sensitivity to NGF-deprivation and die (Bergeron et al. 1998). Other studies have demonstrated a delay in TFD-induced death in sympathetic neurons from caspase-9-null embryos (Deshmukh et al. 2000).

[0006] An understanding of the mechanism of neuronal TFD-induced death which would reconcile these apparently contradictory data has never been achieved.

SUMMARY OF THE INVENTION

[0007] This invention provides a first nucleic acid which specifically hybridizes to a nucleic acid encoding an inhibitor-of-apoptosis protein.

[0008] This invention also provides a composition comprising the first nucleic acid and a carrier.

[0009] This invention further provides a method for inducing a cell's death which comprises contacting the cell with the first nucleic acid under conditions permitting the nucleic acid to enter the cell.

[0010] This invention further provides a method for treating a subject afflicted with cancer which comprises administering to the subject a therapeutically effective amount of the first nucleic acid.

[0011] This invention provides a second nucleic acid that specifically hybridizes to a nucleic acid which encodes a protein, other than caspase-2, that induces cell death.

[0012] This invention further comprises a composition comprising the second nucleic acid and a carrier.

[0013] This invention further provides method for inhibiting a cell's death which comprises contacting the cell with the second nucleic acid under conditions permitting the nucleic acid to enter the cell.

[0014] This invention further provides a method for inhibiting a neuronal cell's death which comprises contacting the cell with the second nucleic acid under conditions permitting the nucleic acid to enter the cell.

[0015] This invention further provides a method for treating a neurodegenerative disorder in a subject which comprises administering to the subject a therapeutically effective amount of the second nucleic acid.

[0016] Finally, this invention provides a method for treating a heart disorder in a subject which comprises administering to the subject a therapeutically effective amount of the second nucleic acid.

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIGS. 1A and 1B

[0018] Differential inhibition of TFD-induced death by DEVD-FMK in wild-type and caspase-2-null neurons. Sympathetic neurons from wild-type (FIG. 1A) and caspase-2-null (FIG. 1B) mice were cultured for five days and then washed and treated with anti-NGF in the presence and absence of BAF (50 μM) or DEVD-FMK (10 μM). Cultures were counted daily and survival is reported relative to that in the same cultures before NGF deprivation and is given as mean±SEM (n=3). Error bars are sometimes too small to be visible. This is a representative experiment. Similar results were obtained in four independent experiments.

[0019]FIG. 2A

[0020]FIG. 2A, like FIGS. 2B-2E, shows differential expression of caspases and caspase regulatory molecules in wild-type and caspase-2-null mice. Relative expression of caspase mRNAs in wild-type and caspase-2-null P1 mouse brains. mRNA was prepared from 6 wild-type and 9 caspase-2-null mouse brains. cDNA from each brain was analyzed individually, using serial dilutions in duplicate, and using real-time PCR. Each sample was analyzed three times. Results were normalized to actin mRNA levels. For each caspase, expression in wild-type brains was set at a value of 1. Data are the means±SEM (n=6 for wild-type, n=9 for caspase-2-null).

[0021]FIG. 2B

[0022] Western blots for caspase-9 and caspase-3. Wild-type and caspase-2-null P1 mouse brains were homogenized in sample buffer and equal amounts of protein (determined by the Bradford protein assay) were subjected to Western blotting using the indicated antisera. Actin staining confirmed equal loading. These are representative blots. Similar results were obtained in 6 independent blots for caspase-9, 3 independent blots for caspase-3.

[0023]FIG. 2C

[0024] Relative expression of Diablo, APAF-1, RAIDD and MIAP3 mRNA in wild-type and caspase-2-null mouse brains. mRNA from 6 wild-type and 9 caspase-2 mouse brains was analyzed using real-time PCR as described in FIG. 2A. Results were normalized to actin mRNA levels. For each transcript, results in wild-type are set at a value of 1. Data are the means±SEM (n=6 for wild-type, n=9 for caspase-2-null).

[0025]FIG. 2D

[0026] Western blots for DIABLO/Smac (also referred to herein as Diablo/SMAC), APAF-1 (also referred to herein as Apaf-1), MIAP3. Wild-type and caspase-2-null P1 mouse brains were homogenized in sample buffer and equal amounts of protein were subjected to Western blotting using the indicated antisera. Actin staining confirmed equal loading. These are representative blots. Similar results were obtained with 6 independent blots for DIABLO/Smac, 3 independent blots for APAF-1 and MIAP3.

[0027]FIG. 2E

[0028] Relative expression of caspase, DIABLO/Smac, and MIAP3 mRNAs in wild-type and caspase-2-null P1 cultured sympathetic neurons. mRNA was prepared from wild-type and caspase-2-null sympatheic neurons grown in culture for 6 days. cDNA was analyzed with real-time PCR using serial dilutions in duplicate. Each sample was analyzed three times. Results were normalized to actin mRNA levels. For each mRNA, expression in wild-type brains was set at a value of 1. Data are the means±SEM (n=3 for wild-type, n=3 for caspase-2-null).

[0029]FIG. 3A

[0030]FIG. 3A, like FIGS. 3B and 3C, shows differential effects of down-regulation of specific caspases on TFD-induced death of wild-type and caspase-2-null sympathetic neurons. PENETRATIN1™-linked antisense oligonucleotides specifically down-regulate targeted caspases. PC12 cells were treated with the indicated antisense oligonucleotides (240 nM) for 6 hours. Cell lysates containing equal amounts of protein were subjected to Western blotting using the corresponding antisera. Actin staining confirmed equal loading. These are representative blots. Similar results were obtained in 2 independent experiments. Sympathetic neurons from P1 wild-type (FIG. 3B) and caspase-2-null (FIG. 3C) mice were cultured for 5 days. Cultures were then washed and treated with anti-NGF in the presence and absence of the indicated antisense oligonucleotides (see legend at right). Cultures were scored daily, and survival is reported relative to the numbers of living neurons in the same cultures before NGF deprivation and is given as mean±SEM (n=3). Error bars are sometimes too small to be visible. This is a representative experiment. Similar results were obtained in 6 independent experiments.

[0031]FIGS. 4A-4E

[0032] Caspase-2-null neurons employ a pathway alternative to TFD-induced death. Sympathetic neurons from P1 wild-type (FIGS. 4A, 4D) and caspase-2-null (FIGS. 4B, 4E) mice were cultured for 5 days. Cultures were then washed and treated with anti-NGF in the presence and absence of V-ADiablo (FIGS. 4A, 4B) or V-AAPAF-1 (FIGS. 4D, 4E). Cultures were scored daily, and survival is reported relative to that in the same cultures before NGF deprivation and is given as mean±SEM (n=3). Error bars are sometimes too small to be visible. This is a representative experiment. Similar results were obtained in 3 independent experiments. Specific down-regulation of DIABLO/Smac and APAF-1 by antisense oligonucleotides is shown in FIG. 4C. PC12 cells were treated with the indicated antisense oligonucleotides (240 nM) for 6 hours. Cells lysates containing equal amounts of protein were subjected to Western blotting using the corresponding antisera. Actin staining confirmed equal loading. These are representative blots. Similar results were obtained in 2 independent experiments.

[0033]FIGS. 5A-5F

[0034] Photomicrographs of SCGs from caspase-2-null mice rescued by down-regulation of various components of the caspase-9 pathway are shown. Sympathetic neurons for caspase-2-null mice were cultured for 5 days. Cultures were then washed and treated with anti-NGF in the presence or absence of various antisense oligonucleotides. The photomicrographs were taken after two days of treatment. FIG. 5A: +NGF; FIG. 5B: Anti-NGF; FIG. 5C: Anti-NGF+V-ADiablo; FIG. 5D: Anti-NGF+V-AAPAF-1; FIG. 5E: Anti-NGF+V-ACasp9; and FIG. 5F: Anti-NGF+V-ACasp3. Bar=100 μm.

[0035]FIGS. 6A-6F

[0036] Down-regulation of various components of the caspase-9 pathway suppresses caspase-3 activation in NGF-deprived sympathetic neurons from caspase-2-null mice. Sympathetic neurons from caspase-2-null mice were cultured on chamber coverglass slides for 5 days. Cultures were then washed and treated with anti-NGF in the presence and absence of various oligonuculeotides. After 5 hours, cells were fixed, immunostained for actin and activated caspase-3 and examined by confocal microscopy (colors not shown). FIG. 6A: +NGF; FIG. 6B: Anti-NGF; FIG. 6C: Anti-NGF+V-ADiablo; FIG. 6D: Anti-NGF+V-AAPAF-1; FIG. 6E: Anti-NGF+V-ACasp9; and FIG. 6F: Anti-NGF+V-ACasp3. Bar=50 μm.

[0037]FIG. 7A

[0038]FIG. 7A, like FIGS. 7B-7E, show that down-regulation of MIAP3 permits caspase-9-dependent TFD-induced death of wild-type SCGs. FIG. 7A shows specific down-regulation of MIAP3. PC12 cells were treated with V-AMIAP3 (240 nM) for 6 hours. Cells lysates containing equal amounts of protein were subjected to Western blotting using the corresponding antisera. Actin staining confirmed equal loading. These are representative blots. Similar results were obtained in 2 independent experiments.

[0039]FIG. 7B

[0040] Sympathetic neurons from wild-type mice were cultured for 5 days. Cultures-were then washed and treated with anti-NGF in the presence and absence of the indicated antisense oligonucleotides. Cultures were scored daily, and survival is reported relative to that in the same cultures before NGF deprivation and is given as mean±SEM (n=3). Error bars are sometimes too small to be visible. This is a representative experiment. Similar results were obtained in 3 independent experiments.

[0041]FIGS. 7C-7E

[0042] Activation of caspase-3 in NGF-deprived sympathetic neurons is dependent on caspase-9. Sympathetic neurons from wild-type mice were cultured on chamber coverglass slides for 5 days.

[0043] Cultures were then washed and treated with anti-NGF in the presence and absence of various oligonucleotides. After 5 hours, cells were fixed, immunostained for actin and activated caspase-3 and examined with confocal microscopy (color not shown). FIG. 7C:-NGF; FIG. 7D: Anti-NGF+V-ACasp2+V-AMIAP3; and FIG. 7E: Anti-NGF+V-ACasp2+V-AMIAP3+V-ACasp9. Bar=50 μm.

[0044]FIGS. 8A and 8B

[0045] Schematic representation of the trophic factor deprivation death pathways in sympathetic neurons.

[0046]FIG. 9

[0047] Relative expression of MIAP mRNAs in wild-type and caspase-2-null P1 cultured sympathetic neurons. mRNA was prepared from wild-type and caspase-2-null sympathetic neurons grown in culture for 6 days. cDNA was analyzed with real-time PCR using serial dilutions in duplicate.

[0048]FIG. 10

[0049] Aβ activation of the caspase-8 pathway is suppressed by MIAP2 in sympathetic neurons. Wild-type sympathetic neuron cultures were treated with aggregated β-amyloid in the presence or absence of the indicated antisense oligonucleotides for 2 days. Cultures were scored daily, and survival is reported relative to the numbers of living neurons in the cultures before NGF deprivation and is given as mean±SEM (n=3).

[0050]FIG. 11

[0051] Aβ death in caspase-2-null SCGs can utilize the caspase-8 pathway when MIAP2 is suppressed. Caspase-2-null sympathetic neuronal cultures were treated with Aβ in the presence or absence of the indicated antisense oligonucleotides for 2 days. Cultures were scored daily, and survival is reported relative to the numbers of living neurons in the same cultures before NGF deprivation and is given as a mean±SEM (n=3).

[0052]FIG. 12

[0053] Aβ activation of the caspase-8 pathway is suppressed by MIAP2 in hippocampal neurons. Cultured hippocampal neurons were treated with Aβ in the presence or absence of the indicated oligonucleotides. Neuronal survival was determined after one day of treatment and is given as a mean±SEM (n=3).

[0054]FIG. 13

[0055] This Figure sets forth the (a) amino acid sequence of, and (b) nucleotide sequence encoding, MIAP1.

[0056]FIG. 14

[0057] This Figure sets forth the (a) amino acid sequence of, and (b) nucleotide sequence encoding, MIAP2.

[0058]FIG. 15

[0059] This Figure sets forth the (a) amino acid sequence of, and (b) nucleotide sequence encoding, MIAP3.

[0060]FIG. 16

[0061] This Figure sets forth the (a) amino acid sequence of, and (b) nucleotide sequence encoding, CIAP1.

[0062]FIG. 17

[0063] This Figure sets forth the (a) amino acid sequence of, and (b) nucleotide sequence encoding, CIAP2.

[0064]FIG. 18

[0065] This Figure sets forth the (a) amino acid sequence of, and (b) nucleotide sequence encoding, XIAP.

[0066]FIG. 19

[0067] This Figure sets forth the amino acid sequence of human Bruce.

[0068]FIG. 20

[0069] This Figure sets forth the (a) amino acid sequence of, and (b) nucleotide sequence encoding, human Survivin.

[0070]FIG. 21

[0071] This Figure sets forth the (a) amino acid sequence of, and (b) nucleotide sequence encoding, human APAF-1.

[0072]FIG. 22

[0073] This Figure sets forth the (a) amino acid sequence of, and (b) nucleotide sequence encoding, human RAIDD.

[0074]FIG. 23

[0075] This Figure sets forth the (a) amino acid sequence of, and (b) nucleotide sequence encoding, human Diablo/SMAC.

[0076]FIG. 24

[0077] This Figure sets forth the amino acid sequence of human NAIP.

DETAILED DESCRIPTION OF THE INVENTION

[0078] Definitions

[0079] “Antibody” shall include, by way of example, both naturally occurring and non-naturally occurring antibodies. Specifically, this term includes polyclonal and monoclonal antibodies, and fragments thereof. Furthermore, this term includes chimeric antibodies and wholly synthetic antibodies, and fragments thereof.

[0080] “Antisense nucleic acid” shall mean any nucleic acid which, when introduced into a cell, specifically hybridizes to at least a portion of an mRNA in the cell encoding a protein (“target protein”) whose expression is to be inhibited, and thereby inhibits the target protein's expression. The instant nucleic acids are antisense nucleic acids, and can hybridize to an mRNA at its protein-coding region and/or its non-coding region (e.g., 5′-untraslated region). Hybridization can also occur at an mRNA splice site, ribosome-binding site, and/or at or near the initiation codon (e.g., from just upstream of the initiation codon to about 10 nucleotides downstream therefrom).

[0081] “Nucleic acid” shall mean any nucleic acid molecule, including, without limitation, DNA, RNA and hybrids thereof. Nucleic acids include, for example, oligonucleotides. The nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art, and are exemplified in PCR Systems, Reagents and Consumables (Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, N.J., USA).

[0082] “Specifically hybridize” to a nucleic acid shall mean, with respect to a first nucleic acid, that the first nucleic acid hybridizes to a second nucleic acid with greater affinity than to any other nucleic acid.

[0083] “Subject” shall mean any animal, such as a human, a non-human primate, a mouse, a rat, a guinea pig or a rabbit.

[0084] “Treating” a disorder shall mean slowing, stopping or reversing the disorder's progression. In the preferred embodiment, treating a disorder means reversing the disorder's progression, ideally to the point of eliminating the disorder itself.

[0085] Embodiments of the Invention

[0086] This invention provides a first nucleic acid which specifically hybridizes to a nucleic acid encoding an inhibitor-of-apoptosis protein.

[0087] In one embodiment, the nucleic acid is complementary to the nucleic acid encoding the inhibitor-of-apoptosis protein. In another embodiment, the nucleic acid is an oligonucleotide having a length of from about 15 nucleotides to about 25 nucleotides. Specifically envisioned is an oligonucleotide having a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. Also envisioned is an oligonucleotide that hybridizes to at least eight consecutive nucleotides, and oligonucleotides of at least about 15 nucleotides in length.

[0088] With respect to the first nucleic acid, the inhibitor-of-apoptosis protein can be any inhibitor-of apoptosis protein including, without limitation, MIAP1 (FIG. 13, GenBank Accession No. NM_(—)007464), MIAP2 (FIG. 14, GenBank Accession No. NM_(—)007465), MIAP3 (FIG. 15, GenBank Accession No. NM009688), CIAP1 (FIG. 16, GenBank Accession No. XM006266), CIAP2 (FIG. 17, GenBank Accession No. XM006267), XIAP (FIG. 18, GenBank Accession No. NM001166, U.S. Pat. Nos. 6,171,821 and 6,159,709), Bruce (FIG. 19), Survivin (FIG. 20, GenBank Accession No. BC008718), NAIP (FIG. 24), ML-IAP and ILP2. In one embodiment, the first nucleic acid specifically hybridizes to the portion of the nucleic acid encoding MIAP3 beginning with the adenosine at position 769 and ending with the guanosine at position 791.

[0089] This invention also provides a first composition comprising the first nucleic acid and a carrier.

[0090] In one embodiment, the first composition comprises nucleic acids which specifically hybridize to nucleic acids encoding a plurality of inhibitor-of-apoptosis proteins. Such pluralities of inhibitor-of-apoptosis proteins include, without limitation, (a) CIAP1, CIAP2 and XIAP; (b) CIAP1 and XIAP; (c) CIAP2 and XIAP; and (d) CIAP1 and CIAP2.

[0091] In one embodiment of the first composition, the carrier comprises a diluent, an adjuvant, a virus, a liposome, a microencapsule, a neuronal cell receptor ligand, a neuronal-specific virus, a polymer-encapsulated cell or a retroviral vector. In another embodiment, the carrier is an aerosol, an intravenous carrier, an oral carrier or a topical carrier.

[0092] This invention further provides a method for inducing a cell's death which comprises contacting the cell with the first nucleic acid under conditions permitting the nucleic acid to enter the cell.

[0093] In one embodiment, this method further comprises contacting the cell with nucleic acids which specifically hybridize to nucleic acids encoding a plurality of inhibitor-of-apoptosis proteins. Each plurality of inhibitor-of-apoptosis proteins includes, without limitation, (a) CIAP1, CIAP2 and XIAP; (b) CIAP1 and XIAP; (c) CIAP2 and XIAP; and (d) CIAP1 and CIAP2.

[0094] In one embodiment of this method, the conditions permitting the nucleic acid to enter the cell comprise the use of a vector, a liposome, a mechanical means or an electrical means. Such vectors include, without limitation, a plasmid, a cosmid, a bacterophage vector, an adenovirus vector, an adeno-associated virus vector, a protein vector (e.g., PENETRATIN1™), an Epstein-Barr virus vector, a Herpes virus vector, an LXSN vector, an LNL6 vector, an attenuated HIV vector (e.g., TAT), a retroviral vector (e.g., MMuLV vector) and a vaccinia virus vector. Such liposomes include, for example, antibody-coated liposomes.

[0095] This invention further provides a method for treating a subject afflicted with cancer which comprises administering to the subject a therapeutically effective amount of the first nucleic acid.

[0096] Cancers treated by this method include, without limitation, acute lymphocytic leukemia, acute myelogenous leukemia, lung cancer, breast cancer, ovarian cancer, prostate cancer, lymphoma, Hodgkin's disease, malignant melanoma, neuroblastoma, renal cell carcinoma and squamous cell carcinoma. In one embodiment, the cancer is a tumor.

[0097] This method can be applied to any subject. In one embodiment, the subject is a mammal. Preferably, the subject is a human.

[0098] This invention provides a second nucleic acid that specifically hybridizes to a nucleic acid which encodes a protein, other than caspase-2, that induces cell death.

[0099] In one embodiment, the nucleic acid is complementary to the nucleic acid encoding the protein that induces cell death. In another embodiment, the nucleic acid is an oligonucleotide having a length of from about 15 nucleotides to about 25 nucleotides. Specifically envisioned is an oligonucleotide having a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. Also envisioned is an oligonucleotide that hybridizes to at least eight consecutive nucleotides, and oligonucleotides of at least about 15 nucleotides in length.

[0100] With respect to the second nucleic acid, the cell death-inducing protein can be any such protein including, without limitation, APAF1 (FIG. 21, GenBank Accession No. AF149794), RAIDD (FIG. 22, GenBank Accession No. U87229, U.S. Pat. No. 6,130,079), Diablo/SMAC (FIG. 23, GenBank Accession No. XM006685), and Htr2/Omi.

[0101] In one embodiment, the second nucleic acid which specifically hybridizes to a nucleic acid encoding the protein APAF1 having the amino acid sequence shown in FIG. 21, and in a further embodiment, specifically hybridizes to the portion of the nucleic acid encoding APAF-1 beginning with the cytosine at position 576 and ending with the adenosine at position 596.

[0102] In another embodiment, the second nucleic acid specifically hybridizes to a nucleic acid encoding the protein RAIDD having the amino acid sequence shown in FIG. 22, and in a further embodiment, specifically hybridizes to the portion of the nucleic acid encoding RAIDD beginning with the guanosine at position 110 and ending with the adenosine at position 130.

[0103] In another embodiment, the second nucleic acid specifically hybridizes to a nucleic acid encoding the protein Diablo/SMAC having the amino acid sequence shown in FIG. 23, and in a further embodiment, specifically hybridizes to the portion of the nucleic acid encoding Diablo/SMAC beginning with the thymidine at position 1 and ending with the thymidine at position 21.

[0104] This invention provides a second composition comprising the second nucleic acid and a carrier. In one embodiment, the second composition comprises nucleic acids which specifically hybridize to nucleic acids encoding a plurality of proteins that induce cell death. Such pluralities include, without limiation, (a) APAF-1 and Diablo/SMAC; (b) APAF-1, Diablo/SMAC and caspase-9; (c) APAF-1, Diablo/SMAC and caspase-7; (d) caspase-2 and RAIDD; (e) caspase-8 and RAIDD; (f) caspase-8, RAIDD and caspase-3; and (g) caspase-2 and caspase-9.

[0105] In one embodiment of the second composition, the carrier comprises a diluent, an adjuvant, a virus, a liposome, a microencapsule, a neuronal cell receptor ligand, a neuronal-specific virus, a polymer-encapsulated cell or a retroviral vector. In another embodiment, the carrier is an aerosol, an intravenous carrier, an oral carrier or a topical carrier.

[0106] This invention further provides a method for inhibiting a cell's death which comprises contacting the cell with the second nucleic acid under conditions permitting the nucleic acid to enter the cell.

[0107] This invention still further provides a method for inhibiting a neuronal cell's death which comprises contacting the cell with the second nucleic acid under conditions permitting the nucleic acid to enter the cell.

[0108] In one embodiment, these methods further comprise contacting the cell with nucleic acids which specifically hybridize to nucleic acids encoding a plurality of proteins that induce cell death. Such pluralities include, without limitation, (a) APAF-1 and Diablo/SMAC; (b) APAF-1, Diablo/SMAC and caspase-9; (c) APAF-1, Diablo/SMAC and caspase-7; (d) caspase-2 and RAIDD; (e) caspase-8 and RAIDD; (f) caspase-8, RAIDD and caspase-3; and (g) caspase-2 and caspase-9.

[0109] In one embodiment of the instant methods, the conditions permitting the second nucleic acid to enter the cell comprise the use of a vector, a liposome, a mechanical means or an electrical means. Such vectors include, without limitation, a plasmid, a cosmid, a bacterophage vector, an adenovirus vector, an adeno-associated virus vector, a protein vector (e.g., PENETRATIN1™), an Epstein-Barr virus vector, a Herpes virus vector, an LXSN vector, an LNL6 vector, an attenuated HIV vector (e.g., TAT), a retroviral vector (e.g. MMuLV vector) and a vaccinia virus vector. Such liposomes include, for example, antibody-coated liposomes.

[0110] This invention further provides a method for treating a neurodegenerative disorder in a subject which comprises administering to the subject a therapeutically effective amount of the second nucleic acid. Neurodegenerative disorders include, for example, brain disorders and central nervous system disorders.

[0111] Finally, this invention provides a method for treating a heart disorder in a subject which comprises administering to the subject a therapeutically effective amount of the second nucleic acid. Heart disorders include, for example, cardiomyopathy.

[0112] The methods employing the second nucleic acid can be applied to any subject. In one embodiment, the subject is a mammal. Preferably, the subject is a human.

[0113] In this invention, nucleic acid sequences encoding certain inhibitor-of-apoptosis proteins and proteins that induce cell death, as well as the amino acid sequences thereof, are set forth herein as follows: MIAP1 (FIG. 13), MIAP2 (FIG. 14), MIAP3 (FIG. 15), CIAP1 (FIG. 16), CIAP2 (FIG. 17), XIAP (FIG. 18), Bruce (FIG. 19), Survivin (FIG. 20), APAF1 (FIG. 21), RAIDD (FIG. 22), and Diablo/SMAC (FIG. 23).

[0114] Determining a therapeutically effective amount of the instant nucleic acids can be done based on animal data using routine computational methods. In one embodiment, the therapeutically effective amount contains between about 0.1 ug and about 1 g of nucleic acid. In other embodiments, the effective amount contains between (a) about 1 ug and about 100 mg of nucleic acid, (b) about 10 ug and about 10 mg of the nucleic acid, (c) about 100 ug and about 1 mg of the nucleic acid, (d) about 1 mg and about 100 mg of the nucleic acid, and (e) about 10 mg and about 50 mg of the nucleic acid. Additionally, the nucleic acid can be administered to the subject, for example, one time only, once in a 24-hour period, more than once in a 24-hour period, and for more than one day.

[0115] In this invention, administering the instant nucleic acids can be effected or performed using any of the various methods and delivery systems known to those skilled in the art. The administering can be performed, for example, intravenously, orally, nasally, via ocular, anal or otic delivery, via implant, via liposome, via viral infection (e.g., via non-integrating, replication-defective virus), via gene bombardment, transmucosally, transdermally, intramuscularly, and subcutaneously. The following delivery systems, which employ a number of routinely used pharmaceutical carriers, are only representative of the many embodiments envisioned for administering the instant compositions.

[0116] Injectable drug delivery systems include solutions, suspensions, gels, microspheres and polymeric injectables, and can comprise excipients such as solubility-altering agents (e.g., ethanol, propylene glycol and sucrose) and polymers (e.g., polycaprylactones and PLGA's). Implantable systems include rods and discs, and can contain excipients such as PLGA and polycaprylactone.

[0117] Oral delivery systems include tablets and capsules. These can contain excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc).

[0118] Transmucosal delivery systems include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid).

[0119] Dermal delivery systems include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer.

[0120] Solutions, suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents (e.g., gums, zanthans, cellulosics and sugars), humectants (e.g., sorbitol), solubilizers (e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservatives and antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid), anti-caking agents, coating agents, and chelating agents (e.g., EDTA).

[0121] This invention will be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.

[0122] Experimental Details

[0123] Evidence suggests that caspases are activated in cascades where upstream (activator) caspases lead to activation of downstream (effector) caspases. One of the most studied cascades is that of caspase-9 which leads to activation of caspases-3 and-7. Activation of the caspase-9 dependent apoptotic pathway is tightly regulated by both the regulatory adaptor molecule Apaf-1, which recruits caspase-9 to the apopotosome, and by the inhibitors of apoptosis proteins (Salvesen 1999; Hengartner 2000). The human IAP, XIAP, has been shown to inhibit caspase-9 as well as the downstream caspases, caspases-3 and -7 (Deveraux et al. 1997). Recent work has revealed a mammalian inhibitor of IAPs, DIABLO/Smac, that inhibits the IAPs and thus promotes caspase-9, -3 and -7 activities (Du et al. 2000; Verhagen et al. 2000). Similar regulation of the caspase-2 pathway has not been found. The death adaptor protein RAIDD activates caspase-2 (Duan and Dixit 1997) but, to date, no IAPs have been found which bind caspase-2 (Deveraux et al. 1999b). The position of caspase-2 in an activation cascade has not been clarified. It has been proposed to act as either an activator or an effector. In any event, it does appear to be independent of the caspase-9 pathway.

[0124] These studies reported reveal specific up-regulation in brains and sympathetic neurons of caspase-2 null mice of both caspase-9 and of DIABLO/Smac. As a consequence of these changes, TFD-induced death, which is normally dependent on caspase-2, switches to an alternative pathway dependent on caspase-9. These results show the tight regulation of caspase activities in neurons. When caspase-2 is removed early in development, as in the null animals, the redundancy of caspases allows the compensations which we have described. The shift from the caspase-2 to the caspase-9 pathway would assure the elimination of neurons which are superfluous. This regulated expression of caspases and IAP inhibitors is likely to be important in neurodegenerative disorders as well as in neurodevelopment.

[0125] Materials and Methods

[0126] Sympathetic Neuron Cultures

[0127] Sympathetic neuron cultures were prepared from 1-day-old wild-type and caspase-2−/− mouse pups (Bergeron et al. 1998). Cultures were grown in 24-well collagen-coated dishes for survival experiments and in 6-well collagen-coated dishes for RNA and protein extraction in RPMI 1640 medium plus 10% horse serum with mouse NGF (100 ng/ml). One day following plating, uridine and 5-fluorodeoxyuridine (10 μM each) were added to the cultures and left for three days to eliminate non-neuronal cells (less than 1% non-neuronal cells remain after 3 days). For survival experiments, on the sixth day following plating, NGF was removed by washing the cultures three times with RPMI 1640 medium plus 10% horse serum, followed by the addition of medium containing anti-mouse NGF (1:200, Sigma). Caspase inhibitors (Enzyme Systems Products) were added as indicated. Each culture was scored, as previously described (Troy et al., 1997), for numbers of living, phase-bright neurons present in the same field at various times. Three replicate cultures were assessed for each condition and data are normalized to numbers of neurons present in each culture at the time of NGF deprivation and reported as mean±SEM. For RNA and protein extraction on the sixth day following plating, RNA and protein were extracted using the Trizol reagent according to the manufacturer's protocol.

[0128] Synthesis of Antisense Oligonucleotides

[0129] Oligonucleotides bearing an SH group at their 5′ end and an NH group at their 3′ end were synthesized by Operon (California). As previously described (Troy et al., 1996a), oligonucleotides were resuspended in deionized water, an equimolar ratio of PENETRATIN1™ (Oncor) was added and the mixture was incubated at 37° C. for 1 hour. The yield of the reaction, estimated by SDS-PAGE followed by Coomassie blue staining, was routinely above 50%. As a control, a scrambled sequence of the antisense oligonucleotide (same base composition, different order) was used. Antisense sequences used were: Acasp1 = CCTCAGGACCTTGTCGGCCAT ACasp3 = GTTGTTGTCCATGGTCACTTT Acasp6 = TGTTTCCATCATGCTTTATTG Acasp7N1 = ATCGTCTGTCATCGTTCCCAC Acasp7N2 = CTCGAAGTCCATACGGTACAG Acasp8 = GTGGAAATCCATTCTTACCAA Acasp9 = CTGCCGGTCCGCCTCGTCCAT Adiablo = AGAGCCGCCATCCCGCGGCCA AAPAF1 = CTTTGCATCCATTGTGCCTCA AMIAP3 = GTTAAAAGTCATCTTCTCTGG

[0130] Western Blotting

[0131] Postnatal day 1 mouse brains were harvested in sample buffer. For antisense down-regulation studies, PC12 cells, grown as previously described, were treated with various antisense constructs for 5 hours and harvested in sample buffer. Equal amounts of protein were separated by 15% PAGE, transferred to nitrocellulose and immunostained as described (Troy et al, 2000). Anti-caspase-9 (MBL) was used at 1:1000, anti-APAF-1 (StressGen) was used at 1:1000, anti-Smac was used at 1:2000, anti-XIAP (StressGen) was used at 1:1000, anti-RAIDD (StressGen) was used at 1:500 and anti-actin (Sigma) was used at 1:200. Visualization was with ECL, using goat-anti-rabbit peroxidase at 1:1000. The relative intensities of the protein bands were quantified using Scion Image 1.55 software (NIH).

[0132] Quantitative PCR

[0133] Primers were designed to amplify a 300-400 base piece of each gene of interest. cDNA from brains of wild-type and caspase-2-null mice or cDNA from cultured sympathetic neurons were added to a reaction mix together with appropriate primers at 0.5 μM each. Reaction mix for the Roche Light Cycler was DNA Master SYBR Green 1 (Roche Molecular Biochemicals). Reaction mix for the Cepheid SMARTCYCLER™, (Fisher) was PCR READY-TO-GO BEADS™ (Amersham Pharmaceuticals) with SYBR GREEN™ (Molecular Probes). Levels of gene transcripts were analyzed using the Roche LIGHT CYCLER™ the Cepheid SMARTCYCLER™ following the manufacturers' specifications. Real time fluorescence of SYBR GREEN™ indicated that double-stranded DNA was measured. Melting curve analysis was used for each protocol to characterize and identify the specific amplicon. In each case quantification was made from the linear portion of the amplification curve. Actin was used to normalize input cDNA.

[0134] Immunocytochemistry

[0135] Sympathetic neurons were grown on.collagen-coated 8 well LabTek chamber slides. After indicated treatments, cells were fixed with 4% paraformaldehyde and immunostained as previously described (Troy et al. 1997). Cells were double labeled with anti-actin (Sigma) at 1:250 and anti-activated caspase-3 (New England Biolabs) at 1:100. Western blotting showed that the lot of the activated caspase-3 antibody used for these studies detected activated caspase-3 but not caspase-3 zymogen. Secondary antibodies were goat-anti-rabbit Alexafluor 546 and goat-anti-mouse Alexafluor 488 (Molecular Probes), both at 1:1000. Cells were examined with a Perkin-Elmer Spinning Disc confocal imaging system mounted on a Nikon inverted microscope.

[0136] Results

[0137] Caspase-2-null Neurons Employ an Alternate Caspase Path to Death after Trophic Factor Deprivation

[0138] Caspase-2 has been identified as critical for trophic factor death in sympathetic neurons and PC12 cells (Troy et al. 1997; Haviv et al. 1998). However, cultured sympathetic neurons from caspase-2-null mice die when deprived of NGF (Bergeron et al. 1998). To ascertain that TFD-induced death in the caspase-2-null neurons was caspase-dependent, both wild-type and caspase-2-null cells were treated with the pseudosubstrate caspase inhibitors BAF and DEVD-FMK. FIG. 1A shows that-the broad-spectrum caspase inhibitor BAF protects caspase-2-null neurons as well as wild-type neurons, confirming that the death process is caspase-mediated in both sets of neurons. However, DEVD-FMK, used at a concentration (10 μM) that is relatively specific for caspase-3 family members, provided protection only for caspase-2-null neurons (FIG. 1B). This suggested that although caspase activity was required for death in both cases after removal of NGF, different caspases were used in each case. The rescue of the caspase-2-null neurons from TFD by DEVD-FMK suggested that this was a member of the caspase-3 family.

[0139] Caspase-2 Null Mice Brains and Sympathetic Neurons have Increased Expression of Caspase-9 and DIABLO/Smac

[0140] The differential effects of DEVD-FMK led us to investigate whether the targeted knock-out of caspase-2 resulted in changes in expression of other caspases or other constituents of cell death pathways that would preserve vulnerability to TFD. Brains, not including cerebella, from wild-type and caspase-2-null postnatal day 1(P1) mice were harvested for RNA and protein, and the relative expression of various caspases was determined using quantitative PCR and Western blotting. These studies revealed that caspase-2 and caspase-9 transcripts are differentially expressed in the two groups of animals; caspase-2-null animals have no caspase-2 mRNA but have more than 3 times the levels of caspase-9 mRNA, relative to actin expression (FIG. 2A). No significant changes were observed for transcripts encoding caspases-1, -3, -6, -7, -8, -11, or -14. The increase in caspase-9 mRNA was confirmed by Northern blotting. Western blotting also revealed changes only in caspase-2 and caspase-9 levels. Caspase-9 protein was increased approximately three-fold in the caspase-2-null mouse brain (FIG. 2B) and, as expected, caspase-2 protein was absent. Other caspases (-1, -3, -6, -7, -8, -11) were unchanged (caspase-3 levels are shown in FIG. 2B). The increase in caspase-9 expression was confirmed in cultured sympathetic neurons as well. Sympathetic neurons from wild-type and caspase-2-null P1 animals were cultured for 5 days and harvested for total cellular RNA and protein assays. Quantitative PCR showed a more than 5-fold increase in caspase-9 expression (FIG. 2E). Western blotting confirmed the increase in caspase-9 protein in the neurons.

[0141] Next, it was investigated whether loss of caspase-2 resulted in changes in other molecules known to modulate the caspase-2 or caspase-9 pathways. These include RAIDD for.caspase-2, and Apaf-1, MIAP3 and DIABLO/Smac for caspase-9. There were no changes in expression of either message or protein for RAIDD, the death adaptor protein for caspase-2 (Duan and Dixit 1997) or Apaf-1, the mammalian ced-4 homologue that activates caspase-9 (Zou et al. 1997) (FIGS. 2C, 2D). However, in the case of the recently discovered DIABLO/Smac, an inhibitor of IAPs (inhibitor of apoptosis proteins) that is permissive for caspase-9 activation (Chai et al. 2000; Du et al. 2000; Verhagen et al. 2000), both mRNA and protein were increased by approximately two-fold in brain, as well as in sympathetic neurons (FIGS. 2C, 2D, 2E). MIAP3, a mouse homologue of XIAP (Farahani et al. 1997), an IAP that has been shown to inhibit the activity of caspases-3, -7 and -9 (Takahashi et al. 1998; Deveraux et al. 1997, 1999a), was unchanged (FIGS. 2C, 2D, 2E).

[0142] Specific Inhibition of the Caspase-9 Pathway Protects Caspase-2-null Neurons but not Wild-type Neurons from TFD

[0143] It was next assessed whether, as indicated by the above findings, TFD-induced death of caspase-2 null and wild-type sympathetic neurons employs different sets of caspases. Since none of the available pharmacologic caspase inhibitors is completely specific for an individual caspase, antisense oligonucleotides were used to decrease expression of specific caspases. This was achieved by using antennapedia peptide (PENETRATIN1™)-mediated intracellular delivery of antisense oligonucleotides, which is a technique that has been widely and successfully used for such purposes (Allinquant et al. 1995; Troy et al. 1996; Pooga et al. 1998; Nakagawa et al. 2000). By this means, 50-80% down-regulation of individual caspases was achieved in cultured neuronal cells without affecting levels of other caspases (Troy et al. 1997; Troy et al. 2000, and representative blots in FIG. 3A). Control (scrambled) oligonucleotides had no discernable effect on expression. Antisense oligonucleotides were designed to down-regulate caspases-1, -2, -3, -6, -7, -8, -9 and linked to PENETRATIN1™. Specificity of sequences was verified by BLAST search (NCBI). Efficacy of down-regulation was evaluated by Western blotting of the target caspase in PC12 cell cultures with or without exposure to the PENETRATIN1™-linked oligonucleotides (FIG. 3A for caspases-6, -7, -8, -9; Troy et al. 1997 for caspase-2; Troy et al. 2000 for caspases-1, -3). PC12 cell cultures were used because of the greater number amount of material available for for the biochemical measurements. Our previous work supports the concurrence of mechanisms in PC12 cells and sympathetic neurons (Farinelli et al. 1996; Park et al. 1998; Stefanis et al. 1998; Troy et al. 1997, 2000). All antisense oligonucleotides provided greater than 50% downregulation of the targeted caspase within 5 hours of treatment. Levels of the other non-targeted caspases were not affected (data not shown). Sympathetic neurons from wild-type and caspase-2 null mice were deprived of NGF in the presence and absence of each of these antisense oligonucleotides and survival assessed daily for three days (FIGS. 3B, 3C). As previously shown, PENETRATIN1™-linked antisense oligonucleotide to caspase-2 (V-ACasp2, previously called V-ANedd) protected wild-type neurons from NGF withdrawal. As anticipated, there was no protection of caspase-2-null neurons by this construct. In contrast, caspase-2-null neurons were protected by V-ACasp3, V-ACasp7 and V-ACasp9. These antisense constructs, however, provided no protection for wild-type neurons. No protection was afforded for either wild-type or caspase-2-null neurons by control (scrambled) oligonucleotides or by downregulation of caspases-1, -6 or -8.

[0144] Downregulation of DIABLO/Smac or Apaf-1 Selectively Protects Caspase-2-null Neurons from TFD

[0145] It was next tested whether down-regulation of additional components of the caspase-9 pathway would bring about differential protection. Antisense-mediated down-regulation of either DIABLO/Smac (FIGS. 4A, 4B) or Apaf-1 (FIGS. 4D, 4E) provided complete protection against NGF withdrawal for caspase-2-null neurons but had no effect on survival of wild-type neurons. FIG. 4C shows the efficacy of down-regulation by these constructs. The photomicrographs in FIGS. 5C-5F show that inhibition of Apaf-1 (FIG. 5C), DIABLO/Smac (Figure 5D), caspase-9 (FIG. 5E) or caspase-3 (FIG. 5F) expression in caspase-2-null neurons protected not only cell bodies, but also neurites.

[0146] Downregulation of DIABLO/Smac, Apaf-1, Caspase-9 or Caspase-3 Suppresses Elevation of Activated Caspase-3 in NGF-deprived Neurons from Caspase-2 null Mice

[0147] The preceding findings point to the activation of.the caspase-9 pathway in caspase-2-null neurons, with consequent activation of caspases-3 and -7. Using an antibody that specifically recognizes activated caspase-3 (see Methods), we assessed the cellular localization of this enzyme in caspase-2-null neurons after various treatments. The confocal micrographs in FIGS. 6A-6F depict cultures of sympathetic neurons from caspase-2-null mice double-labeled for actin (green, but color not shown) and activated caspase-3 (red, but color not shown). Control cells show only minimal staining for activated caspase-3 in either cell bodies and neurites (FIG. 6A). After 5 hours of TFD, there is substantial activation of caspase-3. In the two cells shown in FIG. 6B, it is clear that, as activation of caspase-3 increases, actin immunostaining decreases, likely due to actin degradation during the death process. The induction of activated caspase-3 seen in caspase-2-null neurons after TFD is blocked by downregulation of either Diablo or APAF-1 with the appropriate antisense oligonucleotide. Downregulation of caspase-9 or caspase-3 (FIGS. 6E, 6F) substantially decreased the amount of activated caspase-3 detectable by immunostaining, but did not completely block it.

[0148] The Caspase-9 Pathway is Suppressed in Wild-type Neurons by IAPs

[0149] Although NGF-deprivation induces DEVDase activity in wild-type sympathetic neurons and PC12 cells, this is neither necessary nor sufficient to induce death (Troy et al. 1997; Stefanis et al. 1998). In addition, endogenous suppressors of caspases are likely to play an important role in the regulation of caspase activity and death. The IAP family of caspase inhibitors has been shown to block caspases-3, -7 and -9 activities (Deveraux et al. 1997, 1999b). To reduce IAP activity in cultured sympathetic neurons, a PENETRATIN1™-linked antisense oligonucleotide (V-AMIAP3) to MIAP3 was designed. MIAP3 was chosen because it is the mouse homologue of XIAP, the IAP that has been most closely linked with the caspase-9 pathway. V-AMIAP3 promotes 70% down-regulation of MIAP3 within 5 hours (FIG. 7A). To determine if in vivo activation of the caspase-9 pathway might be suppressed by IAPs in wild-type neurons, we withdrew NGF in the presence of V-ACasp2 and V-AMIAP3. Simultaneous treatment with multiple PENETRATIN1™-linked antisense oligonucleotides does not alter the effects of the individual oligonucleotides (Troy et al. 1996). As shown in FIG. 7B, the protection conferred by caspase-2 down-regulation (by V-ACasp2) was reversed by down-regulation of MIAP3 (by co-treatment with V-AMIAP3). This suggests that reduction of MIAP3 levels permits death by an otherwise suppressed caspase-9-dependent pathway. Consistent with this, the death induced by V-ACasp2 plus V-AMIAP3 was prevented by down-regulation of either Apaf-1 or caspase-9 (FIG. 7B). This suggestion was further supported when we examined immunostaining of activated caspase-3 in these neurons. Withdrawal of NGF in the presence of V-ACasp2plus V-AMIAP3 induced a strong signal in both cell bodies and neurites (FIGS. 7C, 7D). This activated caspase-3 immunostaining was suppressed by the downregulation of caspase-9 (FIG. 7E).

[0150] Discussion

[0151] In an attempt to reconcile the apparently conflicting observations that NGF withdrawal induces apoptosis in caspase-2-null sympathetic neurons (Bergeron et al. 1998), but is unable to cause death in neurons in which caspase-2 has been down regulated (Troy et al. 1997), we examined the ability of caspase inhibitors to rescue each of these cell types from death. Both cell types were rescued by the broad spectrum inhibitor, BAF, but only the caspase-2-null neurons were rescued by DEVD-FMK, which is relatively selective for caspase-3-like activities when used at 10 μM. This differential sensitivity to the caspase inhibitors led us to question whether there were differences in the expression of caspases or the regulators of caspase activity in these two cell types. We found that caspase-9 mRNA and protein are selectively increased by approximately 3-fold in the newborn caspase-2-null mouse brain and more than 5-fold in cultured sympathetic neurons. Expression of the pro-apoptotic death regulator DIABLO/Smac was also elevated.

[0152] The increases in the expression of these two pro-apoptotic molecules suggests that they might compensate for the loss of caspase-2 and enable neurons from caspase-2-null mice to die by an alternative pathway mediated by caspase-9 and its downstream targets such as caspases-3 and -7. This was confirmed in the series of experiments showing that down-regulation of caspase-2 suppresses TFD in wild-type neurons, but not in caspase-2-null neurons. In contrast, down-regulation of caspases-3, -7, and -9 rescues caspase-2-null neurons, but not wild-type neurons, from NGF deprivation. Furthermore, interference with caspase-9 activation by down-regulation of Apaf-1 provided protection for caspase-2-null neurons, but not for wild-type neurons.

[0153] The compensatory switch to the caspase-9 pathway that we observed in caspase-2-null mice appears to involve more than simply elevation of caspase-9 levels. DIABLO/Smac is a recently identified protein that enables activation of caspase-9 (and most likely, caspases-3 and -7) by binding to members of the IAP (inhibitor of apoptosis protein) family (Chai et al. 2000; Du et al. 2000; Verhagen et al. 2000). We observed that DIABLO/Smac levels are doubled in caspase-2-null brains and sympathetic neurons and that down-regulation of DIABLO/Smac protects caspase-2-null, but not wild-type, sympathetic neurons from NGF deprivation. Thus, the availability of the caspase-9 pathway for induction of death in NGF-deprived neurons may be at least in part dependent on its regulation by the competing activities of IAPs and DIABLO/Smac. In caspase-2-null neurons, the elevated levels of DIABLO/Smac might help swing the balance to favor enhanced activation of the caspase-9 pathway.

[0154] The apparent involvement of DIABLO/Smac in promoting death of caspase-2-null neurons raised the issue of whether IAPs may play a role in the repression of the caspase-9 pathway in wild-type neurons. The IAPs effectively suppress activity of caspases-3, -7 and -9 (Deveraux et al. 1997; Deveraux et al. 1998; Deveraux et al. 1999b). We chose to investigate the role of MIAP3, the mouse homologue of XIAP, because it is expressed in sympathetic neurons and can inhibit all three of the above caspases (Farahani et al. 1997). We found that although downregulation of caspase-2 protects wild-type neurons from NGF deprivation, the simultaneous down-regulation of MIAP3 results in death. This death appeared to involve the caspase-9 pathway because it was inhibited in turn by the additional down-regulation of either caspase-9 or Apaf-1. The simplest interpretation of these observations is that when NGF is removed from wild-type neurons in our system, caspase-2 is activated and mediates death, and that the caspase-9 pathway is held in check by MIAP3. When MIAP3 is down-regulated in such neurons, the caspase-9 pathway is no longer suppressed and, when caspase-2 is also down-regulated, the caspase-9 pathway mediates death. A schematic depiction of the alternative death pathways is shown in FIGS. 8A and 8B.

[0155] Taken together, our findings support the idea that wild-type sympathetic neurons possess two alternative caspase pathways that have the potential to mediate TFD-induced death. Under the conditions of our experiments, the caspase-2 pathway is predominant and the caspase-9 pathway is held in check by IAPs. One result of this arrangement is that various circumstances may switch utilization of the two pathways. For instance, as we observed, knockout of caspase-2 results in compensatory enablement of the caspase-9 pathway due, at least in part, to up-regulation of caspase-9 and of DIABLO/Smac. Another variable is developmental stage. Although the existing literature is incomplete, it appears that caspase 9 is highly expressed in the developing mouse brain at embryonic day 7 and declines after that (Kuida et al. 1998).

[0156] In contrast, mouse caspase-2 (originally identified by virtue of its down-regulation in brain during development (Kumar et al. 1994)) is barely expressed at embryonic day 8, and has peak expression in the brain at embryonic day 12. However, rodent sympathetic neurons show high expression of caspase-2 in P1 animals and a subsequent decrease so that expression is minimal by day P11 (Savitz and Kessler 2000). Developmental expression patterns for other elements of either the caspase-2 or caspase-9 pathways have yet to be established in sympathetic neurons, but such time-dependent changes represent potentially important variables in choice of caspase death mechanisms. Similarly, it is likely that additional factors can influence the expression of specific caspases and caspase regulatory molecules, and thereby switch cells from one death pathway to another.

[0157] In light of the aforementioned findings, it is not surprising that circumstances may occur in which TFD-induced death of sympathetic neurons is dependent on the caspase-9 pathway. It was recently reported that sympathetic neurons from caspase-9 null embryos undergo delayed TFD-induced death (Deshmukh et al. 2000) and on this basis, it was suggested that caspase-9 plays a critical role in death caused by NGF deprivation. These studies employed E17 embryos, a developmental stage at which the expression of caspase-9 may normally be higher and caspase- 2 lower than in the postnatal (P1) neurons used in our studies. It is also possible that the knock-out of caspase-9 also leads to depression of the caspase-2 pathway and delay of death.

[0158] The failure of caspase 3 activation to cause death in the wild-type neurons in which caspase-2 has been -down-regulated suggests that the level of activation falls below a critical level for inducing apoptosis. This possibility is supported by the observation that further increasing the activity of the caspase-9 pathway by down-regulation of MIAP3 leads to death and by the increased concentrations of both caspase-9 and DIABLO/Smac in the caspase-2-null mouse brains and neurons. It is possible that “subapoptotic” activation of the caspases in the caspase-9 pathway serves one or more important functions, such as mediating cytoskeletal breakdown.

[0159] In contrast to the caspase 9 pathway, relatively little is known about the mechanisms by which caspase-2 is activated and how such activation leads to death. Our past work indicates that caspase-2 is not downstream of caspase-3-like activity in NGF-deprived sympathetic neurons and visa versa (Stefanis et al. 1998). Caspase-2 possesses a long CARD-containing pro-domain that appears important for activation via specific association with CARD-containing adapter proteins such as RAIDD (Duan and Dixit 1997). However, little is known about how NGF deprivation might trigger interaction between caspase-2 and RAIDD and/or other activators. Several types of evidence indicate that cytochrome C release from mitochondria is required for TFD-induced death of sympathetic neurons (Deshmukh and Johnson 1998; Neame et al. 1998). Given the importance of caspase-2 in TFD-induced death, this raises the yet untested possibility that activation of caspase-2 lies downstream of mitochondrial perturbation. In this regard, it may be relevant that procaspase-2 has been reported to be present within mitochondria and to be released during the apoptotic process (Susin et al. 1999). Also, in contrast to the caspase-9 pathway, there are no currently known negative regulators of caspase-2 activity. Thus it is possible that, unlike caspases -3, -7 and -9 which are subject to inhibition by IAPs, caspase-2, once it is activated, inevitably leads to death.

[0160] The aforementioned findings raise a note of caution regarding interpretation of data from knock-out animals and emphasize the flexibility and redundancy of apoptotic pathways. Two compensatory changes were discovered in expression of apoptosis-related proteins that occur in response to loss of caspase-2 and these appear to contribute to enablement of an alternative apoptotic caspase pathway in sympathetic neurons. It is conceivable that similar or additional changes also occurred in other tissues and that these may affect other aspects of the phenotype of caspase-2-null mice. It has been recently reported that compensatory changes in caspase activation occur in caspase-9 and caspase-3-null animals (Zheng et al. 2000). Although the latter study did not identify specific molecular changes that underlie the compensatory activation of alternative caspases, it does underscore the point raised here that cells possess multiple apoptotic caspase pathways and the means to switch from one to another.

[0161] In summary, the data presented here show that TFD-induced death of sympathetic neurons has the potential to proceed by either of two distinct pathways and that the decision of which pathway is used in a given situation can be regulated by alterations in the relative levels of the components of each of the pathways. It is of particular interest that such regulation included both caspases and an IAP inhibitor. While we have manipulated these levels by genetic and antisense approaches, they are likely regulated to similar effect both during development and in neurodegenerative disorders.

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1 22 1 600 PRT human 1 Met Val Gln Asp Ser Ala Phe Leu Ala Lys Leu Met Lys Ser Ala Asp 1 5 10 15 Thr Phe Glu Leu Lys Tyr Asp Phe Ser Cys Glu Leu Tyr Arg Leu Ser 20 25 30 Thr Tyr Ser Ala Phe Pro Arg Gly Val Pro Val Ser Glu Arg Ser Leu 35 40 45 Ala Arg Ala Gly Phe Tyr Tyr Thr Gly Ala Asn Asp Lys Val Lys Cys 50 55 60 Phe Cys Cys Gly Leu Met Leu Asp Asn Trp Lys Gln Gly Asp Ser Pro 65 70 75 80 Met Glu Lys His Arg Lys Leu Tyr Pro Ser Cys Asn Phe Val Gln Thr 85 90 95 Leu Asn Pro Ala Asn Ser Leu Glu Ala Ser Pro Arg Pro Ser Leu Pro 100 105 110 Ser Thr Ala Met Ser Thr Met Pro Leu Ser Phe Ala Ser Ser Glu Asn 115 120 125 Thr Gly Tyr Phe Ser Gly Ser Tyr Ser Ser Phe Pro Ser Asp Pro Val 130 135 140 Asn Phe Arg Ala Asn Gln Asp Cys Pro Ala Leu Ser Thr Ser Pro Tyr 145 150 155 160 His Phe Ala Met Asn Thr Glu Lys Ala Arg Leu Leu Thr Tyr Glu Thr 165 170 175 Trp Pro Leu Ser Phe Leu Ser Pro Ala Lys Leu Ala Lys Ala Gly Phe 180 185 190 Tyr Tyr Ile Gly Pro Gly Asp Arg Val Ala Cys Phe Ala Cys Asp Gly 195 200 205 Lys Leu Ser Asn Trp Glu Arg Lys Asp Asp Ala Met Ser Glu His Gln 210 215 220 Arg His Phe Pro Ser Cys Pro Phe Leu Lys Asp Leu Gly Gln Ser Ala 225 230 235 240 Ser Arg Tyr Thr Val Ser Asn Leu Ser Met Gln Thr His Ala Ala Arg 245 250 255 Ile Arg Thr Phe Ser Asn Trp Pro Ser Ser Ala Leu Val His Ser Gln 260 265 270 Glu Leu Ala Ser Ala Gly Phe Tyr Tyr Thr Gly His Ser Asp Asp Val 275 280 285 Lys Cys Phe Cys Cys Asp Gly Gly Leu Arg Cys Trp Glu Ser Gly Asp 290 295 300 Asp Pro Trp Val Glu His Ala Lys Trp Phe Pro Arg Cys Glu Tyr Leu 305 310 315 320 Leu Arg Ile Lys Gly Gln Glu Phe Val Ser Gln Val Gln Ala Gly Tyr 325 330 335 Pro His Leu Leu Glu Gln Leu Leu Ser Thr Ser Asp Ser Pro Glu Asp 340 345 350 Glu Asn Ala Asp Ala Ala Ile Val His Phe Gly Pro Gly Glu Ser Ser 355 360 365 Glu Asp Val Val Met Met Ser Thr Pro Val Val Lys Ala Ala Leu Glu 370 375 380 Met Gly Phe Ser Arg Ser Leu Val Arg Gln Thr Val Gln Arg Gln Ile 385 390 395 400 Leu Ala Thr Gly Glu Asn Tyr Arg Thr Val Ser Asp Leu Val Ile Gly 405 410 415 Leu Leu Asp Ala Glu Asp Glu Met Arg Glu Glu Gln Met Glu Gln Ala 420 425 430 Ala Glu Glu Glu Glu Ser Asp Asp Leu Ala Leu Ile Arg Lys Asn Lys 435 440 445 Met Val Leu Phe Gln His Leu Thr Cys Val Thr Pro Met Leu Tyr Cys 450 455 460 Leu Leu Ser Ala Arg Ala Ile Thr Glu Gln Glu Cys Asn Ala Val Lys 465 470 475 480 Gln Lys Pro His Thr Leu Gln Ala Ser Thr Leu Ile Asp Thr Val Leu 485 490 495 Ala Lys Gly Asn Thr Ala Ala Thr Ser Phe Arg Asn Ser Leu Arg Glu 500 505 510 Ile Asp Pro Ala Leu Tyr Arg Asp Ile Phe Val Gln Gln Asp Ile Arg 515 520 525 Ser Leu Pro Thr Asp Asp Ile Ala Ala Leu Pro Met Glu Glu Gln Leu 530 535 540 Arg Lys Leu Gln Glu Glu Arg Met Cys Lys Val Cys Met Asp Arg Glu 545 550 555 560 Val Ser Ile Val Phe Ile Pro Cys Gly His Leu Val Val Cys Lys Asp 565 570 575 Cys Ala Pro Ser Leu Arg Lys Cys Pro Ile Cys Arg Gly Thr Ile Lys 580 585 590 Gly Thr Val Arg Thr Phe Leu Ser 595 600 2 2673 DNA human 2 tgggagttcc ccggagccct ggaggaaagc accgcaggtc tgagcagccc tgagccgggc 60 agggtggggg cagtggctaa ggcctagctg gggacgattt aaaggtatcg cgccacccag 120 ccacacccca caggccaggc gagggtgcca cccccggaga tcagaggtca ttgctggcgt 180 tcagagccta ggaagtgggc tgcggtatca gcctagcagt aaaaccgacc agaagccatg 240 cacaaaacta catccccaga gaaagacttg tcccttcccc tccctgtcat ctcaccatga 300 acatggttca agacagcgcc tttctagcca agctgatgaa gagtgctgac acctttgagt 360 tgaagtatga cttttcctgt gagctgtacc gattgtccac gtattcagct tttcccaggg 420 gagttcctgt gtcagaaagg agtctggctc gtgctggctt ttactacact ggtgccaatg 480 acaaggtcaa gtgcttctgc tgtggcctga tgctagacaa ctggaaacaa ggggacagtc 540 ccatggagaa gcacagaaag ttgtacccca gctgcaactt tgtacagact ttgaatccag 600 ccaacagtct ggaagctagt cctcggcctt ctcttccttc cacggcgatg agcaccatgc 660 ctttgagctt tgcaagttct gagaatactg gctatttcag tggctcttac tcgagctttc 720 cctcagaccc tgtgaacttc cgagcaaatc aagattgtcc tgctttgagc acaagtccct 780 accactttgc aatgaacaca gagaaggcca gattactcac ctatgaaaca tggccattgt 840 cttttctgtc accagcaaag ctggccaaag caggcttcta ctacatagga cctggagata 900 gagtggcctg ctttgcgtgc gatgggaaac tgagcaactg ggaacgtaag gatgatgcta 960 tgtcagagca ccagaggcat ttccccagct gtccgttctt aaaagacttg ggtcagtctg 1020 cttcgagata cactgtctct aacctgagca tgcagacaca cgcagcccgt attagaacat 1080 tctctaactg gccttctagt gcactagttc attcccagga acttgcaagt gcgggctttt 1140 attatacagg acacagtgat gatgtcaagt gtttttgctg tgatggtggg ctgaggtgct 1200 gggaatctgg agatgacccc tgggtggaac atgccaagtg gtttccaagg tgtgagtact 1260 tgctcagaat caaaggccaa gaatttgtca gccaagttca agctggctat cctcatctac 1320 ttgagcagct attatctacg tcagactccc cagaagatga gaatgcagac gcagcaatcg 1380 tgcattttgg ccctggagaa agttcggaag atgtcgtcat gatgagcacg cctgtggtta 1440 aagcagcctt ggaaatgggc ttcagtagga gcctggtgag acagacggtt cagcggcaga 1500 tcctggccac tggtgagaac tacaggaccg tcagtgacct cgttataggc ttactcgatg 1560 cagaagacga gatgagagag gagcagatgg agcaggcggc cgaggaggag gagtcagatg 1620 atctagcact aatccggaag aacaaaatgg tgcttttcca acatttgacg tgtgtgacac 1680 caatgctgta ttgcctccta agtgcaaggg ccatcactga acaggagtgc aatgctgtga 1740 aacagaaacc acacacctta caagcaagca cactgattga tactgtgtta gcaaaaggaa 1800 acactgcagc aacctcattc agaaactccc ttcgggaaat tgaccctgcg ttatacagag 1860 atatatttgt gcaacaggac attaggagtc ttcccacaga tgacattgca gctctaccaa 1920 tggaagaaca gttgcggaaa ctccaggagg aaagaatgtg taaagtgtgt atggaccgag 1980 aggtatccat cgtgttcatt ccctgtggcc atctggtcgt gtgcaaagac tgcgctccct 2040 ctctgaggaa gtgtcccatc tgtagaggga ccatcaaggg cacagtgcgc acatttctct 2100 cctgaacaag actaatggtc catggctgca acttcagcca ggaggaagtt cactgtcact 2160 cccagctcca ttcggaactt gaggccagcc tggatagcac gagacaccgc caaacacaca 2220 aatataaaca tgaaaaactt ttgtctgaag tcaagaatga atgaattact tatataataa 2280 ttttaattgg tttccttaaa agtgctattt gttcccaact cagaaaattg ttttctgtaa 2340 acatatttac atactacctg catctaaagt attcatatat tcatatattc agatgtcatg 2400 agagagggtt ttgttcttgt tcctgaaaag cagggattgc ctgcactcct gaaattctca 2460 gaaagattta caatgttggc atttatggtt cagaaactag aatcttctcc cgttgcttta 2520 agaaccggga gcacagatgt ccatgtgttt tatgtataga aattcctgtt atttattgga 2580 tgacatttta gggatatgaa atttttataa agaatttgtg agaaaaagtt aataaagcaa 2640 cataattacc tctttttttt taaagaaaaa aaa 2673 3 612 PRT human 3 Met Asp Lys Thr Val Ser Gln Arg Leu Gly Gln Gly Thr Leu His Gln 1 5 10 15 Lys Leu Lys Arg Ile Met Glu Lys Ser Thr Ile Leu Ser Asn Trp Thr 20 25 30 Lys Glu Ser Glu Glu Lys Met Lys Phe Asp Phe Ser Cys Glu Leu Tyr 35 40 45 Arg Met Ser Thr Tyr Ser Ala Phe Pro Arg Gly Val Pro Val Ser Glu 50 55 60 Arg Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly Val Asn Asp Lys 65 70 75 80 Val Lys Cys Phe Cys Cys Gly Leu Met Leu Asp Asn Trp Lys Gln Gly 85 90 95 Asp Ser Pro Val Glu Lys His Arg Gln Phe Tyr Pro Ser Cys Ser Phe 100 105 110 Val Gln Thr Leu Leu Ser Ala Ser Leu Gln Ser Pro Ser Lys Asn Met 115 120 125 Ser Pro Val Lys Ser Arg Phe Ala His Ser Ser Pro Leu Glu Arg Gly 130 135 140 Gly Ile His Ser Asn Leu Cys Ser Ser Pro Leu Asn Ser Arg Ala Val 145 150 155 160 Glu Asp Phe Ser Ser Arg Met Asp Pro Cys Ser Tyr Ala Met Ser Thr 165 170 175 Glu Glu Ala Arg Phe Leu Thr Tyr Ser Met Trp Pro Leu Ser Phe Leu 180 185 190 Ser Pro Ala Glu Leu Ala Arg Ala Gly Phe Tyr Tyr Ile Gly Pro Gly 195 200 205 Asp Arg Val Ala Cys Phe Ala Cys Gly Gly Lys Leu Ser Asn Trp Glu 210 215 220 Pro Lys Asp Asp Ala Met Ser Glu His Arg Arg His Phe Pro His Cys 225 230 235 240 Pro Phe Leu Glu Asn Thr Ser Glu Thr Gln Arg Phe Ser Ile Ser Asn 245 250 255 Leu Ser Met Gln Thr His Ser Ala Arg Leu Arg Thr Phe Leu Tyr Trp 260 265 270 Pro Pro Ser Val Pro Val Gln Pro Glu Gln Leu Ala Ser Ala Gly Phe 275 280 285 Tyr Tyr Val Asp Arg Asn Asp Asp Val Lys Cys Phe Cys Cys Asp Gly 290 295 300 Gly Leu Arg Cys Trp Glu Pro Gly Asp Asp Pro Trp Ile Glu His Ala 305 310 315 320 Lys Trp Phe Pro Arg Cys Glu Phe Leu Ile Arg Met Lys Gly Gln Glu 325 330 335 Phe Val Asp Glu Ile Gln Ala Arg Tyr Pro His Leu Leu Glu Gln Leu 340 345 350 Leu Ser Thr Ser Asp Thr Pro Gly Glu Glu Asn Ala Asp Pro Thr Glu 355 360 365 Thr Val Val His Phe Gly Pro Gly Glu Ser Ser Lys Asp Val Val Met 370 375 380 Met Ser Thr Pro Val Val Lys Ala Ala Leu Glu Met Gly Phe Ser Arg 385 390 395 400 Ser Leu Val Arg Gln Thr Val Gln Arg Gln Ile Leu Ala Thr Gly Glu 405 410 415 Asn Tyr Arg Thr Val Asn Asp Ile Val Ser Val Leu Leu Asn Ala Glu 420 425 430 Asp Glu Arg Arg Glu Glu Glu Lys Glu Arg Gln Thr Glu Glu Met Ala 435 440 445 Ser Gly Asp Leu Ser Leu Ile Arg Lys Asn Arg Met Ala Leu Phe Gln 450 455 460 Gln Leu Thr His Val Leu Pro Ile Leu Asp Asn Leu Leu Glu Ala Ser 465 470 475 480 Val Ile Thr Lys Gln Glu His Asp Ile Ile Arg Gln Lys Thr Gln Ile 485 490 495 Pro Leu Gln Ala Arg Glu Leu Ile Asp Thr Val Leu Val Lys Gly Asn 500 505 510 Ala Ala Ala Asn Ile Phe Lys Asn Ser Leu Lys Glu Ile Asp Ser Thr 515 520 525 Leu Tyr Glu Asn Leu Phe Val Glu Lys Asn Met Lys Tyr Ile Pro Thr 530 535 540 Glu Asp Val Ser Gly Leu Ser Leu Glu Glu Gln Leu Arg Arg Leu Gln 545 550 555 560 Glu Glu Arg Thr Cys Lys Val Cys Met Asp Arg Glu Val Ser Ile Val 565 570 575 Phe Ile Pro Cys Gly His Leu Val Val Cys Gln Glu Cys Ala Pro Ser 580 585 590 Leu Arg Lys Cys Pro Ile Cys Arg Gly Thr Ile Lys Gly Thr Val Arg 595 600 605 Thr Phe Leu Ser 610 4 3151 DNA human 4 agttatataa aatacgaagt tttcaaaaag aaggctagtg caacagaaaa gctttgctaa 60 aacagattct tagttatttg aggtaacaaa agaaagccat gtcttgaatt gattcgttct 120 taattataac agacttatag tggaaagggc cttaaacaca ggcggacttt ataaaatgca 180 gtcttaggtt tatgtgcaaa atactgtctg ttgaccagat gtattcacat gatatataca 240 gagtcaaggt ggtgatatag aagatttaac agtgagggag ttaacagtct gtgctttaag 300 cgcagttcct ttacagtgaa tactgtagtc ttaatagacc tgagctgact gctgcagttg 360 atgtaaccca ctttagagaa tactgtatga catcttctct aaggaaaacc agctgcagac 420 ttcactcagt tcctttcatt tcataggaaa aggagtagtt cagatgtcat gtttaagtcc 480 ttataaggga aaagagcctg aatatatgcc ctagtaccta ggcttcataa ctagtaataa 540 gaagttagtt atgggtaaat agatctcagg ttacccagaa gagttcatgt gacccccaaa 600 gagtcctaac tagtgtcttg gcaagtgaga cagatttgtc ctgtgagggt gtcaattcac 660 cagtccaagc agaagacaat gaatctatcc agtcaggtgt ctgtggtgga gatctagtgt 720 ccaagtggtg agaaacttca tctggaagtt taagcggtca gaaatactat tactactcat 780 ggacaaaact gtctcccaga gactcggcca aggtacctta caccaaaaac ttaaacgtat 840 aatggagaag agcacaatct tgtcaaattg gacaaaggag agcgaagaaa aaatgaagtt 900 tgacttttcg tgtgaactct accgaatgtc tacatattca gcttttccca ggggagttcc 960 tgtctcagag aggagtctgg ctcgtgctgg cttttattat acaggtgtga atgacaaagt 1020 caagtgcttc tgctgtggcc tgatgttgga taactggaaa caaggggaca gtcctgttga 1080 aaagcacaga cagttctatc ccagctgcag ctttgtacag actctgcttt cagccagtct 1140 gcagtctcca tctaagaata tgtctcctgt gaaaagtaga tttgcacatt cgtcacctct 1200 ggaacgaggt ggcattcact ccaacctgtg ctctagccct cttaattcta gagcagtgga 1260 agacttctca tcaaggatgg atccctgcag ctatgccatg agtacagaag aggccagatt 1320 tcttacttac agtatgtggc ctttaagttt tctgtcacca gcagagctgg ccagagctgg 1380 cttctattac atagggcctg gagacagggt ggcctgtttt gcctgtggtg ggaaactgag 1440 caactgggaa ccaaaggatg atgctatgtc agagcaccgc agacattttc cccactgtcc 1500 atttctggaa aatacttcag aaacacagag gtttagtata tcaaatctaa gtatgcagac 1560 acactctgct cgattgagga catttctgta ctggccacct agtgttcctg ttcagcccga 1620 gcagcttgca agtgctggat tctattacgt ggatcgcaat gatgatgtca agtgcttttg 1680 ttgtgatggt ggcttgagat gttgggaacc tggagatgac ccctggatag aacacgccaa 1740 atggtttcca aggtgtgagt tcttgatacg gatgaagggt caggagtttg ttgatgagat 1800 tcaagctaga tatcctcatc ttcttgagca gctgttgtcc acttcagaca ccccaggaga 1860 agaaaatgct gaccctacag agacagtggt gcattttggc cctggagaaa gttcgaaaga 1920 tgtcgtcatg atgagcacgc ctgtggttaa agcagccttg gaaatgggct tcagtaggag 1980 cctggtgaga cagacggttc agcggcagat cctggccact ggtgagaact acaggaccgt 2040 caatgatatt gtctcagtac ttttgaatgc tgaagatgag agaagagaag aggagaagga 2100 aagacagact gaagagatgg catcaggtga cttatcactg attcggaaga atagaatggc 2160 cctctttcaa cagttgacac atgtccttcc tatcctggat aatcttcttg aggccagtgt 2220 aattacaaaa caggaacatg atattattag acagaaaaca cagataccct tacaagcaag 2280 agagcttatt gacaccgttt tagtcaaggg aaatgctgca gccaacatct tcaaaaactc 2340 tctgaaggaa attgactcca cgttatatga aaacttattt gtggaaaaga atatgaagta 2400 tattccaaca gaagacgttt caggcttgtc attggaagag cagttgcgga gattacaaga 2460 agaacgaact tgcaaagtgt gtatggacag agaggtttct attgtgttca ttccgtgtgg 2520 tcatctagta gtctgccagg aatgtgcccc ttctctaagg aagtgcccca tctgcagggg 2580 gacaatcaag gggactgtgc gcacatttct ctcatgagtg aagaatggtc tgaaagtatt 2640 gttggacatc agaagctgtc agaacaaaga atgaactact gatttcagct cttcagcagg 2700 acattctact ctctttcaag attagtaatc ttgctttatg aagggtagca ttgtatattt 2760 aagcttagtc tgttgcaagg gaaggtctat gctgttgagc tacaggactg tgtctgttcc 2820 agagcaggag ttgggatgct tgctgtatgt ccttcaggac ttcttggatt tggaatttgt 2880 gaaagctttg gattcaggtg atgtggagct cagaaatcct gaaaccagtg gctctggtac 2940 tcagtagtta gggtaccctg tgcttcttgg tgcttttcct ttctggaaaa taaggatttt 3000 tctgctactg gtaaatattt tctgtttgtg agaaatatat taaagtgttt cttttaaagg 3060 cgtgcatcat tgtagtgtgt gcagggatgt atgcaggcaa aacactgtgt atataataaa 3120 taaatctttt taaaaagtgt aaaaaaaaaa a 3151 5 496 PRT human 5 Met Thr Phe Asn Ser Phe Glu Gly Thr Arg Thr Phe Val Leu Ala Asp 1 5 10 15 Thr Asn Lys Asp Glu Glu Phe Val Glu Glu Phe Asn Arg Leu Lys Thr 20 25 30 Phe Ala Asn Phe Pro Ser Ser Ser Pro Val Ser Ala Ser Thr Leu Ala 35 40 45 Arg Ala Gly Phe Leu Tyr Thr Gly Glu Gly Asp Thr Val Gln Cys Phe 50 55 60 Ser Cys His Ala Ala Ile Asp Arg Trp Gln Tyr Gly Asp Ser Ala Val 65 70 75 80 Gly Arg His Arg Arg Ile Ser Pro Asn Cys Arg Phe Ile Asn Gly Phe 85 90 95 Tyr Phe Glu Asn Gly Ala Ala Gln Ser Thr Asn Pro Gly Ile Gln Asn 100 105 110 Gly Gln Tyr Lys Ser Glu Asn Cys Val Gly Asn Arg Asn Pro Phe Ala 115 120 125 Pro Asp Arg Pro Pro Glu Thr His Ala Asp Tyr Leu Leu Arg Thr Gly 130 135 140 Gln Val Val Asp Ile Ser Asp Thr Ile Tyr Pro Arg Asn Pro Ala Met 145 150 155 160 Cys Ser Glu Glu Ala Arg Leu Lys Ser Phe Gln Asn Trp Pro Asp Tyr 165 170 175 Ala His Leu Thr Pro Arg Glu Leu Ala Ser Ala Gly Leu Tyr Tyr Thr 180 185 190 Gly Ala Asp Asp Gln Val Gln Cys Phe Cys Cys Gly Gly Lys Leu Lys 195 200 205 Asn Trp Glu Pro Cys Asp Arg Ala Trp Ser Glu His Arg Arg His Phe 210 215 220 Pro Asn Cys Phe Phe Val Leu Gly Arg Asn Val Asn Val Arg Ser Glu 225 230 235 240 Ser Gly Val Ser Ser Asp Arg Asn Phe Pro Asn Ser Thr Asn Ser Pro 245 250 255 Arg Asn Pro Ala Met Ala Glu Tyr Glu Ala Arg Ile Val Thr Phe Gly 260 265 270 Thr Trp Thr Ser Ser Val Asn Lys Glu Gln Leu Ala Arg Ala Gly Phe 275 280 285 Tyr Ala Leu Gly Glu Gly Asp Lys Val Lys Cys Phe His Cys Gly Gly 290 295 300 Gly Leu Thr Asp Trp Lys Pro Ser Glu Asp Pro Trp Asp Gln His Ala 305 310 315 320 Lys Cys Tyr Pro Gly Cys Lys Tyr Leu Leu Asp Glu Lys Gly Gln Glu 325 330 335 Tyr Ile Asn Asn Ile His Leu Thr His Pro Leu Glu Glu Ser Leu Gly 340 345 350 Arg Thr Ala Glu Lys Thr Pro Pro Leu Thr Lys Lys Ile Asp Asp Thr 355 360 365 Ile Phe Gln Asn Pro Met Val Gln Glu Ala Ile Arg Met Gly Phe Ser 370 375 380 Phe Lys Asp Leu Lys Lys Thr Met Glu Glu Lys Ile Gln Thr Ser Gly 385 390 395 400 Ser Ser Tyr Leu Ser Leu Glu Val Leu Ile Ala Asp Leu Val Ser Ala 405 410 415 Gln Lys Asp Asn Thr Glu Asp Glu Ser Ser Gln Thr Ser Leu Gln Lys 420 425 430 Asp Ile Ser Thr Glu Glu Gln Leu Arg Arg Leu Gln Glu Glu Lys Leu 435 440 445 Ser Lys Ile Cys Met Asp Arg Asn Ile Ala Ile Val Phe Phe Pro Cys 450 455 460 Gly His Leu Ala Thr Cys Lys Gln Cys Ala Glu Ala Val Asp Lys Cys 465 470 475 480 Pro Met Cys Tyr Thr Val Ile Thr Phe Asn Gln Lys Ile Phe Met Ser 485 490 495 6 2691 DNA human 6 attttttaaa ttgatgcatt aacattctaa acattcatct gtttttaaat agtaaaaatt 60 gaactttgcc ttgaatatgt aatgattcat tataacaatt atgcatagtc tttaataatc 120 tgcatatttt atgctgcttt catgtttttc ctaattaatg acttcacatg tttaatattt 180 ataatttttc tgtcatagtt tccatattta tataaaatga atacttaaga tcagtaattc 240 tgctctgttt gtttatatac tattttccat caaaagacaa aatgggactg aggttgaggc 300 tcgttgctaa agcactttcc taaaatgcaa aaggccctat gatggatccc tagtacttat 360 ttaagtgaga gagaaacagg ctgggggtgt aggtctgtta gagcatgtgt ttggcattat 420 gtgaagccca aacactaaaa aaggagaaca aacaaaagcg cagactttaa aactcaagtg 480 gtttggtaat gtacgactct actgtttaga attaaaatgt gtcttagtta ttgtgccatt 540 atttttatgt catcactgga taatatatta gtgcttagta tcagaaatag tccttatgct 600 ttgtgttttg aagttcctaa tgcaatgttc tctttctaga aaaggtggac aagtcctatt 660 ttccagagaa gatgactttt aacagttttg aaggaactag aacttttgta cttgcagaca 720 ccaataagga tgaagaattt gtagaagagt ttaatagatt aaaaacattt gctaacttcc 780 caagtagtag tcctgtttca gcatcaacat tggcgcgagc tgggtttctt tataccggtg 840 aaggagacac cgtgcaatgt ttcagttgtc atgcggcaat agatagatgg cagtatggag 900 actcagctgt tggaagacac aggagaatat ccccaaattg cagatttatc aatggttttt 960 attttgaaaa tggtgctgca cagtctacaa atcctggtat ccaaaatggc cagtacaaat 1020 ctgaaaactg tgtgggaaat agaaatcctt ttgcccctga caggccacct gagactcatg 1080 ctgattatct cttgagaact ggacaggttg tagatatttc agacaccata tacccgagga 1140 accctgccat gtgtagtgaa gaagccagat tgaagtcatt tcagaactgg ccggactatg 1200 ctcatttaac ccccagagag ttagctagtg ctggcctcta ctacacaggg gctgatgatc 1260 aagtgcaatg cttttgttgt gggggaaaac tgaaaaattg ggaaccctgt gatcgtgcct 1320 ggtcagaaca caggagacac tttcccaatt gcttttttgt tttgggccgg aacgttaatg 1380 ttcgaagtga atctggtgtg agttctgata ggaatttccc aaattcaaca aactctccaa 1440 gaaatccagc catggcagaa tatgaagcac ggatcgttac ttttggaaca tggacatcct 1500 cagttaacaa ggagcagctt gcaagagctg gattttatgc tttaggtgaa ggcgataaag 1560 tgaagtgctt ccactgtgga ggagggctca cggattggaa gccaagtgaa gacccctggg 1620 accagcatgc taagtgctac ccagggtgca aatacctatt ggatgagaag gggcaagaat 1680 atataaataa tattcattta acccatccac ttgaggaatc tttgggaaga actgctgaaa 1740 aaacaccacc gctaactaaa aaaatcgatg ataccatctt ccagaatcct atggtgcaag 1800 aagctatacg aatgggattt agcttcaagg accttaagaa aacaatggaa gaaaaaatcc 1860 aaacatccgg gagcagctat ctatcacttg aggtcctgat tgcagatctt gtgagtgctc 1920 agaaagataa tacggaggat gagtcaagtc aaacttcatt gcagaaagac attagtactg 1980 aagagcagct aaggcgccta caagaggaga agctttccaa aatctgtatg gatagaaata 2040 ttgctatcgt tttttttcct tgtggacatc tggccacttg taaacagtgt gcagaagcag 2100 ttgacaaatg tcccatgtgc tacaccgtca ttacgttcaa ccaaaaaatt tttatgtctt 2160 agtggggcac cacatgttat gttcttcttg ctctaattga atgtgtaatg ggagcgaact 2220 ttaagtaatc ctgcatttgc attccattag catcctgctg tttccaaatg gagaccaatg 2280 ctaacagcac tgtttccgtc taaacattca atttctggat ctttcgagtt atcagctgta 2340 tcatttagcc agtgttttac tcgattgaaa ccttagacag agaagcattt tatagctttt 2400 cacatgtata ttggtagtac actgacttga tttctatatg taagtgaatt catcacctgc 2460 atgtttcatg ccttttgcat aagcttaaca aatggagtgt tctgtataag catggagatg 2520 tgatggaatc tgcccaatga ctttaattgg cttattgtaa acacggaaag aactgcccca 2580 cgctgctggg aggataaaga ttgttttaga tgctcacttc tgtgttttag gattctgccc 2640 atttacttgg aatttattgg agttataatg tacttatatg atatttccga a 2691 7 459 PRT human 7 Met His Lys Thr Ala Ser Gln Arg Leu Phe Pro Gly Pro Ser Tyr Gln 1 5 10 15 Asn Ile Lys Ser Ile Met Glu Asp Ser Thr Ile Leu Ser Asp Trp Thr 20 25 30 Asn Ser Asn Lys Gln Lys Met Lys Tyr Asp Phe Ser Cys Glu Leu Tyr 35 40 45 Arg Met Ser Thr Tyr Ser Thr Phe Pro Ala Gly Val Pro Val Ser Glu 50 55 60 Arg Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly Val Asn Asp Lys 65 70 75 80 Val Lys Cys Phe Cys Cys Gly Leu Met Leu Asp Asn Trp Lys Leu Gly 85 90 95 Asp Ser Pro Ile Gln Lys His Lys Gln Leu Tyr Pro Ser Cys Ser Phe 100 105 110 Ile Gln Asn Leu Val Ser Ala Ser Leu Gly Ser Thr Ser Lys Asn Thr 115 120 125 Ser Pro Met Arg Asn Ser Phe Ala His Ser Leu Ser Pro Thr Leu Glu 130 135 140 His Ser Ser Leu Phe Ser Gly Ser Tyr Ser Ser Leu Ser Pro Asn Pro 145 150 155 160 Leu Asn Ser Arg Ala Val Glu Asp Ile Ser Ser Ser Arg Thr Asn Pro 165 170 175 Tyr Ser Tyr Ala Met Ser Thr Glu Glu Ala Arg Phe Leu Thr Tyr His 180 185 190 Met Trp Pro Leu Thr Phe Leu Ser Pro Ser Glu Leu Ala Arg Ala Gly 195 200 205 Phe Tyr Tyr Ile Gly Pro Gly Asp Arg Val Ala Cys Phe Ala Cys Gly 210 215 220 Gly Lys Leu Ser Asn Trp Glu Pro Lys Asp Asp Ala Met Ser Glu His 225 230 235 240 Arg Arg His Phe Pro Asn Cys Pro Phe Leu Glu Asn Ser Leu Glu Thr 245 250 255 Leu Arg Phe Ser Ile Ser Asn Leu Ser Met Gln Thr His Ala Ala Arg 260 265 270 Met Arg Thr Phe Met Tyr Trp Pro Ser Ser Val Pro Val Gln Pro Glu 275 280 285 Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Gly Arg Asn Asp Asp Val 290 295 300 Lys Cys Phe Cys Cys Asp Gly Gly Leu Arg Cys Trp Glu Ser Gly Asp 305 310 315 320 Asp Pro Trp Val Glu His Ala Lys Trp Phe Pro Arg Cys Glu Phe Leu 325 330 335 Ile Arg Met Lys Gly Gln Glu Phe Val Asp Glu Ile Gln Gly Arg Tyr 340 345 350 Pro His Leu Leu Glu Gln Leu Leu Ser Thr Ser Asp Thr Thr Gly Glu 355 360 365 Glu Asn Ala Asp Pro Pro Ile Ile His Phe Gly Pro Gly Glu Ser Ser 370 375 380 Ser Glu Asp Ala Val Met Met Asn Thr Pro Val Val Lys Ser Ala Leu 385 390 395 400 Glu Met Gly Phe Asn Arg Asp Leu Val Lys Gln Thr Val Gln Ser Lys 405 410 415 Ile Leu Thr Thr Gly Glu Asn Tyr Lys Thr Val Asn Asp Ile Val Ser 420 425 430 Ala Leu Leu Asn Ala Glu Asp Glu Lys Arg Glu Glu Glu Lys Glu Lys 435 440 445 Gln Ala Glu Glu Met Ala Ser Val Ile Cys His 450 455 8 3495 DNA human 8 gaattctatg gagtgtaatt ttgtgtatga attatatttt taaaacattg aagagttttc 60 agaaagaagg ctagtagagt tgattactga tactttatgc taagcagtac ttttttggta 120 gtacaatatt ttgttaggcg tttctgataa cactagaaag gacaagtttt atcttgtgat 180 aaattgatta atgtttacaa catgactgat aattatagct gaatagtcct taaatgatga 240 acaggttatt tagtttttaa atgcagtgta aaaagtgtgc tgtggaaatt ttatggctaa 300 ctaagtttat ggagaaaata ccttcagttg atcaagaata atagtggtat acaaagttag 360 gaagaaagtc aacatgatgc tgcaggaaat ggaaacaaat acaaatgata tttaacaaag 420 atagagttta cagtttttga actttaagcc aaattcattt gacatcaagc actatagcag 480 gcacaggttc aacaaagctt gtgggtattg acttccccca aaagttgtca gctgaagtaa 540 tttagcccac ttaagtaaat actatgatga taagctgtgt gaacttagct tttaaatagt 600 gtgaccatat gaaggtttta attacttttg tttattggaa taaaatgaga ttttttgggt 660 tgtcatgtta aagtgcttat agggaaagaa gcctgcatat aattttttac cttgtggcat 720 aatcagtaat tggtctgtta ttcaggcttc atagcttgta accaaatata aataaaaggc 780 ataatttagg tattctatag ttgcttagaa ttttgttaat ataaatctct gtgaaaaatc 840 aaggagtttt aatattttca gaagtgcatc cacctttcag ggctttaagt tagtattact 900 caagattatg aacaaatagc acttaggtta cctgaaagag ttactacaac cccaaagagt 960 tgtgttctaa gtagtatctt ggtaattcag agagatactc atcctacctg aatataaact 1020 gagataaatc cagtaaagaa agtgtagtaa attctacata agagtctatc attgatttct 1080 ttttgtggta aaaatcttag ttcatgtgaa gaaatttcat gtgaatgttt tagctatcaa 1140 acagtactgt cacctactca tgcacaaaac tgcctcccaa agacttttcc caggtccctc 1200 gtatcaaaac attaagagta taatggaaga tagcacgatc ttgtcagatt ggacaaacag 1260 caacaaacaa aaaatgaagt atgacttttc ctgtgaactc tacagaatgt ctacatattc 1320 aactttcccc gccggggtgc ctgtctcaga aaggagtctt gctcgtgctg gtttttatta 1380 tactggtgtg aatgacaagg tcaaatgctt ctgttgtggc ctgatgctgg ataactggaa 1440 actaggagac agtcctattc aaaagcataa acagctatat cctagctgta gctttattca 1500 gaatctggtt tcagctagtc tgggatccac ctctaagaat acgtctccaa tgagaaacag 1560 ttttgcacat tcattatctc ccaccttgga acatagtagc ttgttcagtg gttcttactc 1620 cagcctttct ccaaaccctc ttaattctag agcagttgaa gacatctctt catcgaggac 1680 taacccctac agttatgcaa tgagtactga agaagccaga tttcttacct accatatgtg 1740 gccattaact tttttgtcac catcagaatt ggcaagagct ggtttttatt atataggacc 1800 tggagatagg gtagcctgct ttgcctgtgg tgggaagctc agtaactggg aaccaaagga 1860 tgatgctatg tcagaacacc ggaggcattt tcccaactgt ccatttttgg aaaattctct 1920 agaaactctg aggtttagca tttcaaatct gagcatgcag acacatgcag ctcgaatgag 1980 aacatttatg tactggccat ctagtgttcc agttcagcct gagcagcttg caagtgctgg 2040 tttttattat gtgggtcgca atgatgatgt caaatgcttt tgttgtgatg gtggcttgag 2100 gtgttgggaa tctggagatg atccatgggt agaacatgcc aagtggtttc caaggtgtga 2160 gttcttgata cgaatgaaag gccaagagtt tgttgatgag attcaaggta gatatcctca 2220 tcttcttgaa cagctgttgt caacttcaga taccactgga gaagaaaatg ctgacccacc 2280 aattattcat tttggacctg gagaaagttc ttcagaagat gctgtcatga tgaatacacc 2340 tgtggttaaa tctgccttgg aaatgggctt taatagagac ctggtgaaac aaacagttca 2400 aagtaaaatc ctgacaactg gagagaacta taaaacagtt aatgatattg tgtcagcact 2460 tcttaatgct gaagatgaaa aaagagaaga ggagaaggaa aaacaagctg aagaaatggc 2520 atcagtgatt tgtcattaat tcggaagaac agaatggctc tctttcaaca attgacatgt 2580 gtgcttccta tcctggataa tcttttaaag gccaatgtaa ttaataaaca ggaacatgat 2640 attattaaac aaaaaacaca gataccttta caagcgagag aactgattga taccattttg 2700 gttaaaggaa atgctgcggc caacatcttc aaaaactgtc taaaagaaat tgactctaca 2760 ttgtataaga acttatttgt ggataagaat atgaagtata ttccaacaga agatgtttca 2820 ggtctgtcac tggaagaaca attgaggagg ttgcaagaag aacgaacttg taaagtgtgt 2880 atggacaaag aagtttctgt tgtatttatt ccttgtggtc atctggtagt atgccaggaa 2940 tgtgcccctt ctctaagaaa atgccctatt tgcaggggta taatcaaggg tactgttcgt 3000 acatttctct cttaaagaaa aatagtctat attttaacct gcataaaaag gtctttaaaa 3060 tattgttgaa cacttgaagc catctaaagt aaaaagggaa ttatgagttt ttcaattagt 3120 aacattcatg ttctagtctg ctttggtact aataatcttg tttctgaaaa gatggtatca 3180 tatatttaat cttaatctgt ttatttacaa gggaagattt atgtttggtg aactatatta 3240 gtatgtatgt gtacctaagg gagtagtgtc actgcttgtt atgcatcatt tcaggagtta 3300 ctggatttgt tgttctttca gaaagctttg aatactaaat tatagtgtag aaaagaactg 3360 gaaaccagga actctggagt tcatcagagt tatggtgccg aattgtcttt ggtgcttttc 3420 acttgtgttt taaaataagg atttttctct tatttctccc cctagtttgt gagaaacatc 3480 tcaataaagt gcttt 3495 9 557 PRT human 9 Met Asn Ile Val Glu Asn Ser Ile Phe Leu Ser Asn Leu Met Lys Ser 1 5 10 15 Ala Asn Thr Phe Glu Leu Lys Tyr Asp Leu Ser Cys Glu Leu Tyr Arg 20 25 30 Met Ser Thr Tyr Ser Thr Phe Pro Ala Gly Val Pro Val Ser Glu Arg 35 40 45 Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly Val Asn Asp Lys Val 50 55 60 Lys Cys Phe Cys Cys Gly Leu Met Leu Asp Asn Trp Lys Arg Gly Asp 65 70 75 80 Ser Pro Thr Glu Lys His Lys Lys Leu Tyr Pro Ser Cys Arg Phe Val 85 90 95 Gln Ser Leu Asn Ser Val Asn Asn Leu Glu Ala Thr Ser Gln Pro Thr 100 105 110 Phe Pro Ser Ser Val Thr Asn Ser Thr His Ser Leu Leu Pro Gly Thr 115 120 125 Glu Asn Ser Gly Tyr Phe Arg Gly Ser Tyr Ser Asn Ser Pro Ser Asn 130 135 140 Pro Val Asn Ser Arg Ala Asn Gln Asp Phe Ser Ala Leu Met Arg Ser 145 150 155 160 Ser Tyr His Cys Ala Met Asn Asn Glu Asn Ala Arg Leu Leu Thr Phe 165 170 175 Gln Thr Trp Pro Leu Thr Phe Leu Ser Pro Thr Asp Leu Ala Lys Ala 180 185 190 Gly Phe Tyr Tyr Ile Gly Pro Gly Asp Arg Val Ala Cys Phe Ala Cys 195 200 205 Gly Gly Lys Leu Ser Asn Trp Glu Pro Lys Asp Asn Ala Met Ser Glu 210 215 220 His Leu Arg His Phe Pro Lys Cys Pro Phe Ile Glu Asn Gln Leu Gln 225 230 235 240 Asp Thr Ser Arg Tyr Thr Val Ser Asn Leu Ser Met Gln Thr His Ala 245 250 255 Ala Arg Phe Lys Thr Phe Phe Asn Trp Pro Ser Ser Val Leu Val Asn 260 265 270 Pro Glu Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Gly Asn Ser Asp 275 280 285 Asp Val Lys Cys Phe Cys Cys Asp Gly Gly Leu Arg Cys Trp Glu Ser 290 295 300 Gly Asp Asp Pro Trp Val Gln His Ala Lys Trp Phe Pro Arg Cys Glu 305 310 315 320 Tyr Leu Ile Arg Ile Lys Gly Gln Glu Phe Ile Arg Gln Val Gln Ala 325 330 335 Ser Tyr Pro His Leu Leu Glu Gln Leu Leu Ser Thr Ser Asp Ser Pro 340 345 350 Gly Asp Glu Asn Ala Glu Ser Ser Ile Ile His Phe Glu Pro Gly Glu 355 360 365 Asp His Ser Glu Asp Ala Ile Met Met Asn Thr Pro Val Ile Asn Ala 370 375 380 Ala Val Glu Met Gly Phe Ser Arg Ser Leu Val Lys Gln Thr Val Gln 385 390 395 400 Arg Lys Ile Leu Ala Thr Gly Glu Asn Tyr Arg Leu Val Asn Asp Leu 405 410 415 Val Leu Asp Leu Leu Asn Ala Glu Asp Glu Ile Arg Glu Glu Glu Arg 420 425 430 Glu Arg Ala Thr Glu Glu Lys Glu Ser Asn Asp Leu Leu Leu Ile Arg 435 440 445 Lys Asn Arg Met Ala Leu Phe Gln His Leu Thr Cys Val Ile Pro Ile 450 455 460 Leu Asp Ser Leu Leu Thr Ala Gly Ile Ile Asn Glu Gln Glu His Asp 465 470 475 480 Val Ile Lys Gln Lys Thr Gln Thr Ser Leu Gln Ala Arg Glu Leu Ile 485 490 495 Asp Thr Ile Leu Val Lys Gly Asn Ile Ala Ala Thr Val Phe Arg Asn 500 505 510 Ser Leu Gln Glu Ala Glu Ala Val Leu Tyr Glu His Leu Phe Val Gln 515 520 525 Gln Asp Ile Lys Tyr Ile Pro Thr Glu Asp Val Ser Gly Ser Thr Ser 530 535 540 Gly Arg Thr Ile Ala Glu Thr Thr Arg Arg Lys Asn Met 545 550 555 10 3153 DNA human 10 gaattcaaaa tgtcttcagt tgtaaatctt accattattt tacgtacctc taagaaataa 60 aagtgcttct aattaaaata tgatgtcatt aattatgaaa tacttcttga taacagaagt 120 tttaaaatag ccatcttaga atcagtgaaa tatggtaatg tattattttc ctcctttgag 180 ttaggtcttg tgcttttttt tcctggccac taaatttcac aatttccaaa aagcaaaata 240 aacatattct gaatattttt gctgtgaaac acttgacagc agagctttcc accatgaaaa 300 gaagcttcat gagtcacaca ttacatcttt gggttgattg aatgccactg aaacattcta 360 gtagcctgga gaagttgacc tacctgtgga gatgcctgcc attaaatggc atcctgatgg 420 cttaatacac atcactcttc tgtgaagggt tttaattttc aacacagctt actctgtagc 480 atcatgttta cattgtatgt ataaagatta tacaaaggtg caattgtgta tttcttcctt 540 aaaatgtatc agtataggat ttagaatctc catgttgaaa ctctaaatgc atagaaataa 600 aaataataaa aaatttttca ttttggcttt tcagcctagt attaaaactg ataaaagcaa 660 agccatgcac aaaactacct ccctagagaa aggctagtcc cttttcttcc ccattcattt 720 cattatgaac atagtagaaa acagcatatt cttatcaaat ttgatgaaaa gcgccaacac 780 gtttgaactg aaatacgact tgtcatgtga actgtaccga atgtctacgt attccacttt 840 tcctgctggg gttcctgtct cagaaaggag tcttgctcgt gctggtttct attacactgg 900 tgtgaatgac aaggtcaaat gcttctgttg tggcctgatg ctggataact ggaaaagagg 960 agacagtcct actgaaaagc ataaaaagtt gtatcctagc tgcagattcg ttcagagtct 1020 aaattccgtt aacaacttgg aagctacctc tcagcctact tttccttctt cagtaacaaa 1080 ttccacacac tcattacttc cgggtacaga aaacagtgga tatttccgtg gctcttattc 1140 aaactctcca tcaaatcctg taaactccag agcaaatcaa gatttttctg ccttgatgag 1200 aagttcctac cactgtgcaa tgaataacga aaatgccaga ttacttactt ttcagacatg 1260 gccattgact tttctgtcgc caacagatct ggcaaaagca ggcttttact acataggacc 1320 tggagacaga gtggcttgct ttgcctgtgg tggaaaattg agcaattggg aaccgaagga 1380 taatgctatg tcagaacacc tgagacattt tcccaaatgc ccatttatag aaaatcagct 1440 tcaagacact tcaagataca cagtttctaa tctgagcatg cagacacatg cagcccgctt 1500 taaaacattc tttaactggc cctctagtgt tctagttaat cctgagcagc ttgcaagtgc 1560 gggtttttat tatgtgggta acagtgatga tgtcaaatgc ttttgctgtg atggtggact 1620 caggtgttgg gaatctggag atgatccatg ggttcaacat gccaagtggt ttccaaggtg 1680 tgagtacttg ataagaatta aaggacagga gttcatccgt caagttcaag ccagttaccc 1740 tcatctactt gaacagctgc tatccacatc agacagccca ggagatgaaa atgcagagtc 1800 atcaattatc cattttgaac ctggagaaga ccattcagaa gatgcaatca tgatgaatac 1860 tcctgtgatt aatgctgccg tggaaatggg ctttagtaga agcctggtaa aacagacagt 1920 tcagagaaaa atcctagcaa ctggagagaa ttatagacta gtcaatgatc ttgtgttaga 1980 cttactcaat gcagaagatg aaataaggga agaggagaga gaaagagcaa ctgaggaaaa 2040 agaatcaaat gatttattat taatccggaa gaatagaatg gcactttttc aacatttgac 2100 ttgtgtaatt ccaatcctgg atagtctact aactgccgga attattaatg aacaagaaca 2160 tgatgttatt aaacagaaga cacagacgtc tttacaagca agagaactga ttgatacgat 2220 tttagtaaaa ggaaatattg cagccactgt attcagaaac tctctgcaag aagctgaagc 2280 tgtgttatat gagcatttat ttgtgcaaca ggacataaaa tatattccca cagaagatgt 2340 ttcaggatct accagtggaa gaacaattgc ggagactaca agaagaaaga acatgtaaag 2400 tgtgtatgga caaagaagtg tccatagtgt ttattccttg tggtcatcta gtagtatgca 2460 aagattgtgc tccttcttta agaaagtgtc ctatttgtag gagtacaatc aagggtacag 2520 ttcgtacatt tctttcatga agaagaacca aaacatcgtc taaactttag aattaattta 2580 ttaaatgtat tataacttta acttttatcc taatttggtt tccttaaaat ttttatttat 2640 ttacaactca aaaaacattg ttttgtgtaa catatttata tatgtatcta aaccatatga 2700 acatatattt tttagaaact aagagaatga taggcttttg ttcttatgaa cgaaaaagag 2760 gtagcactac aaacacaata ttcaatcaaa atttcagcat tattgaaatt gtaagtgaag 2820 taaaacttaa gatatttgag ttaaccttta agaattttaa atattttggc attgtactaa 2880 taccgggaac atgaagccag gtgtggtggt atgtgcctgt agtcccaggc tgaggcaaga 2940 gaattacttg agcccaggag tttgaatcca tcctgggcag catactgaga ccctgccttt 3000 aaaaacaaac agaacaaaaa caaaacacca gggacacatt tctctgtctt ttttgatcag 3060 tgtcctatac atcgaaggtg tgcatatatg ttgaatgaca ttttagggac atggtgtttt 3120 tataaagaat tctgtgagaa aaaatttaat aaa 3153 11 618 PRT human 11 Met His Lys Thr Ala Ser Gln Arg Leu Phe Pro Gly Pro Ser Tyr Gln 1 5 10 15 Asn Ile Lys Ser Ile Met Glu Asp Ser Thr Ile Leu Ser Asp Trp Thr 20 25 30 Asn Ser Asn Lys Gln Lys Met Lys Tyr Asp Phe Ser Cys Glu Leu Tyr 35 40 45 Arg Met Ser Thr Tyr Ser Thr Phe Pro Ala Gly Val Pro Val Ser Glu 50 55 60 Arg Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly Val Asn Asp Lys 65 70 75 80 Val Lys Cys Phe Cys Cys Gly Leu Met Leu Asp Asn Trp Lys Leu Gly 85 90 95 Asp Ser Pro Ile Gln Lys His Lys Gln Leu Tyr Pro Ser Cys Ser Phe 100 105 110 Ile Gln Asn Leu Val Ser Ala Ser Leu Gly Ser Thr Ser Lys Asn Thr 115 120 125 Ser Pro Met Arg Asn Ser Phe Ala His Ser Leu Ser Pro Thr Leu Glu 130 135 140 His Ser Ser Leu Phe Ser Gly Ser Tyr Ser Ser Leu Ser Pro Asn Pro 145 150 155 160 Leu Asn Ser Arg Ala Val Glu Asp Ile Ser Ser Ser Arg Thr Asn Pro 165 170 175 Tyr Ser Tyr Ala Met Ser Thr Glu Glu Ala Arg Phe Leu Thr Tyr His 180 185 190 Met Trp Pro Leu Thr Phe Leu Ser Pro Ser Glu Leu Ala Arg Ala Gly 195 200 205 Phe Tyr Tyr Ile Gly Pro Gly Asp Arg Val Ala Cys Phe Ala Cys Gly 210 215 220 Gly Lys Leu Ser Asn Trp Glu Pro Lys Asp Asp Ala Met Ser Glu His 225 230 235 240 Arg Arg His Phe Pro Asn Cys Pro Phe Leu Glu Asn Ser Leu Glu Thr 245 250 255 Leu Arg Phe Ser Ile Ser Asn Leu Ser Met Gln Thr His Ala Ala Arg 260 265 270 Met Arg Thr Phe Met Tyr Trp Pro Ser Ser Val Pro Val Gln Pro Glu 275 280 285 Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Gly Arg Asn Asp Asp Val 290 295 300 Lys Cys Phe Cys Cys Asp Gly Gly Leu Arg Cys Trp Glu Ser Gly Asp 305 310 315 320 Asp Pro Trp Val Glu His Ala Lys Trp Phe Pro Arg Cys Glu Phe Leu 325 330 335 Ile Arg Met Lys Gly Gln Glu Phe Val Asp Glu Ile Gln Gly Arg Tyr 340 345 350 Pro His Leu Leu Glu Gln Leu Leu Ser Thr Ser Asp Thr Thr Gly Glu 355 360 365 Glu Asn Ala Asp Pro Pro Ile Ile His Phe Gly Pro Gly Glu Ser Ser 370 375 380 Ser Glu Asp Ala Val Met Met Asn Thr Pro Val Val Lys Ser Ala Leu 385 390 395 400 Glu Met Gly Phe Asn Arg Asp Leu Val Lys Gln Thr Val Gln Ser Lys 405 410 415 Ile Leu Thr Thr Gly Glu Asn Tyr Lys Thr Val Asn Asp Ile Val Ser 420 425 430 Ala Leu Leu Asn Ala Glu Asp Glu Lys Arg Glu Glu Glu Lys Glu Lys 435 440 445 Gln Ala Glu Glu Met Ala Ser Asp Asp Leu Ser Leu Ile Arg Lys Asn 450 455 460 Arg Met Ala Leu Phe Gln Gln Leu Thr Cys Val Leu Pro Ile Leu Asp 465 470 475 480 Asn Leu Leu Lys Ala Asn Val Ile Asn Lys Gln Glu His Asp Ile Ile 485 490 495 Lys Gln Lys Thr Gln Ile Pro Leu Gln Ala Arg Glu Leu Ile Asp Thr 500 505 510 Ile Leu Val Lys Gly Asn Ala Ala Ala Asn Ile Phe Lys Asn Cys Leu 515 520 525 Lys Glu Ile Asp Ser Thr Leu Tyr Lys Asn Leu Phe Val Asp Lys Asn 530 535 540 Met Lys Tyr Ile Pro Thr Glu Asp Val Ser Gly Leu Ser Leu Glu Glu 545 550 555 560 Gln Leu Arg Arg Leu Gln Glu Glu Arg Thr Cys Lys Val Cys Met Asp 565 570 575 Lys Glu Val Ser Val Val Phe Ile Pro Cys Gly His Leu Val Val Cys 580 585 590 Gln Glu Cys Ala Pro Ser Leu Arg Lys Cys Pro Ile Cys Arg Gly Ile 595 600 605 Ile Lys Gly Thr Val Arg Thr Phe Leu Ser 610 615 12 3495 DNA human 12 gaattctatg gagtgtaatt ttgtgtatga attatatttt taaaacattg aagagttttc 60 agaaagaagg ctagtagagt tgattactga tactttatgc taagcagtac ttttttggta 120 gtacaatatt ttgttaggcg tttctgataa cactagaaag gacaagtttt atcttgtgat 180 aaattgatta atgtttacaa catgactgat aattatagct gaatagtcct taaatgatga 240 acaggttatt tagtttttaa atgcagtgta aaaagtgtgc tgtggaaatt ttatggctaa 300 ctaagtttat ggagaaaata ccttcagttg atcaagaata atagtggtat acaaagttag 360 gaagaaagtc aacatgatgc tgcaggaaat ggaaacaaat acaaatgata tttaacaaag 420 atagagttta cagtttttga actttaagcc aaattcattt gacatcaagc actatagcag 480 gcacaggttc aacaaagctt tgggtattga cttcccccaa aagttgtcag ctgaagtaat 540 ttagcccact taagtaaata ctatgatgat aagctgtgtg aacttagctt ttaaatagtg 600 tgaccatatg aaggttttaa ttacttttgt ttattggaat aaaatgagat tttttgggtt 660 gtcatgttaa agtgcttata gggaaagaag cctgcatata attttttacc ttgtggcata 720 atcagtaatt ggtctgttat tcaggcttca tagcttgtaa ccaaatataa ataaaaggca 780 taatttaggt attctatagt tgcttagaat tttgttaata taaatctctg tgaaaaatca 840 aggagtttta atattttcag aagtgcatcc acctttcagg gctttaagtt agtattactc 900 aagattatga acaaatagca cttaggttac ctgaaagagt tactacaacc ccaaagagtt 960 gtgttctaag tagtatcttg gaaattcaga gagatactca tcctacctga atataaactg 1020 agataaatcc agtaaagaaa gtgtagtaaa ttctacataa gagtctatca ttgatttctt 1080 ttggtggtaa aaatcttagt tcatgtgaag aaatttcatg tgaatgtttt agctatcaaa 1140 cagcactgtc acctactcat gcacaaaact gcctcccaaa gacttttccc aggtccctcg 1200 tatcaaaaca ttaagagtat aatggaagat agcacgatct tgtcagattg gacaaacagc 1260 aacaaacaaa aaatgaagta tgacttttcc tgtgaactct acagaatgtc tacatattca 1320 actttccccg ccggggtgcc tgtctcagaa aggagtcttg ctcgtgctgg tttttattat 1380 actggtgtga atgacaaggt caaatgcttc tgttgtggcc tgatgctgga taactggaaa 1440 ctaggagaca gtcctattca aaagcataaa cagctatatc ctagctgtag ctttattcag 1500 aatctggttt cagctagtct gggatccacc tctaagaata cgtctccaat gagaaacagt 1560 tttgcacatt cattatctcc caccttggaa catagtagct tgttcagtgg ttcttactcc 1620 agcctttctc caaaccctct taattctaga gcagttgaag acatctcttc atcgaggact 1680 aacccctaca gttatgcaat gagtactgaa gaagccagat ttcttaccta ccatatgtgg 1740 ccattaactt ttttgtcacc atcagaattg gcaagagctg gtttttatta tataggacct 1800 ggagataggg tagcctgctt tgcctgtggt gggaagctca gtaactggga accaaaggat 1860 gatgctatgt cagaacaccg gaggcatttt cccaactgtc catttttgga aaattctcta 1920 gaaactctga ggtttagcat ttcaaatctg agcatgcaga cacatgcagc tcgaatgaga 1980 acatttatgt actggccatc tagtgttcca gttcagcctg agcagcttgc aagtgctggt 2040 ttttattatg tgggtcgcaa tgatgatgtc aaatgctttt gttgtgatgg tggcttgagg 2100 tgttgggaat ctggagatga tccatgggta gaacatgcca agtggtttcc aaggtgtgag 2160 ttcttgatac gaatgaaagg ccaagagttt gttgatgaga ttcaaggtag atatcctcat 2220 cttcttgaac agctgttgtc aacttcagat accactggag aagaaaatgc tgacccacca 2280 attattcatt ttggacctgg agaaagttct tcagaagatg ctgtcatgat gaatacacct 2340 gtggttaaat ctgccttgga aatgggcttt aatagagacc tggtgaaaca aacagttcaa 2400 agtaaaatcc tgacaactgg agagaactat aaaacagtta atgatattgt gtcagcactt 2460 ctaaatgctg aagatgaaaa aagagaggag gagaaggaaa aacaagctga agaaatggca 2520 tcagatgatt tgtcattaat tcggaagaac agaatggctc tctttcaaca attgacatgt 2580 gtgcttccta tcctggataa tcttttaaag gccaatgtaa ttaataaaca ggaacatgat 2640 attattaaac aaaaaacaca gataccttta caagcgagag aactgattga taccattttg 2700 gttaaaggaa atgctgcggc caacatcttc aaaaactgtc taaaagaaat tgactctaca 2760 ttgtataaga acttatttgt ggataagaat atgaagtata ttccaacaga agatgtttca 2820 ggtctgtcac tggaagaaca attgaggagg ttgcaagaag aacgaacttg taaagtgtgt 2880 atggacaaag aagtttctgt tgtatttatt ccttgtggtc atctggtagt atgccaggaa 2940 tgtgcccctt ctctaagaaa atgccctatt tgcaggggta taatcaaggg tactgttcgt 3000 acatttctct cttaaagaaa aatagtctat attttaacct gcataaaaag gtctttaaaa 3060 tattgttgaa cacttgaagc catctaaagt aaaaagggaa ttatgagttt ttcaattagt 3120 aacattcatg ttctagtctg ctttggtact aataatcttg tttctgaaaa gatggtatca 3180 tatatttaat cttaatctgt ttatttacaa gggaagattt atgtttggtg aactatatta 3240 gtatgtatgt gtacctaagg gagtagtgtc actgcttgtt atgcatcatt tcaggagtta 3300 ctggatttgt tgttctttca gaaagctttg aatactaaat tatagtgtag aaaagaactg 3360 gaaaccagga actctggagt tcatcagagt tatggtgccg aattgtcttt ggtgcttttc 3420 acttgtgttt taaaataagg atttttctct tatttctccc cctagtttgt gagaaacatc 3480 tcaataaagt gcttt 3495 13 806 PRT human 13 Met Ala Ser Gly Ala Asp Ser Lys Gly Asp Asp Leu Ser Thr Ala Ile 1 5 10 15 Leu Lys Gln Lys Asn Arg Pro Asn Arg Leu Ile Val Asp Glu Ala Ile 20 25 30 Asn Glu Asp Asn Ser Val Val Ser Leu Ser Gln Pro Lys Met Asp Glu 35 40 45 Leu Gln Leu Phe Arg Gly Asp Thr Val Leu Leu Lys Gly Lys Lys Arg 50 55 60 Arg Glu Ala Val Cys Ile Val Leu Ser Asp Asp Thr Cys Ser Asp Glu 65 70 75 80 Lys Ile Arg Met Asn Arg Val Val Arg Asn Asn Leu Arg Val Arg Leu 85 90 95 Gly Asp Val Ile Ser Ile Gln Pro Cys Pro Asp Val Lys Tyr Gly Lys 100 105 110 Arg Ile His Val Leu Pro Ile Asp Asp Thr Val Glu Gly Ile Thr Gly 115 120 125 Asn Leu Phe Glu Val Tyr Leu Lys Pro Tyr Phe Leu Glu Ala Tyr Arg 130 135 140 Pro Ile Arg Lys Gly Asp Ile Phe Leu Val Arg Gly Gly Met Arg Ala 145 150 155 160 Val Glu Phe Lys Val Val Glu Thr Asp Pro Ser Pro Tyr Cys Ile Val 165 170 175 Ala Pro Asp Thr Val Ile His Cys Glu Gly Glu Pro Ile Lys Arg Glu 180 185 190 Asp Glu Glu Glu Ser Leu Asn Glu Val Gly Tyr Asp Asp Ile Gly Gly 195 200 205 Cys Arg Lys Gln Leu Ala Gln Ile Lys Glu Met Val Glu Leu Pro Leu 210 215 220 Arg His Pro Ala Leu Phe Lys Ala Ile Gly Val Lys Pro Pro Arg Gly 225 230 235 240 Ile Leu Leu Tyr Gly Pro Pro Gly Thr Gly Lys Thr Leu Ile Ala Arg 245 250 255 Ala Val Ala Asn Glu Thr Gly Ala Phe Phe Phe Leu Ile Asn Gly Pro 260 265 270 Glu Ile Met Ser Lys Leu Ala Gly Glu Ser Glu Ser Asn Leu Arg Lys 275 280 285 Ala Phe Glu Glu Ala Glu Lys Asn Ala Pro Ala Ile Ile Phe Ile Asp 290 295 300 Glu Leu Asp Ala Ile Ala Pro Lys Arg Glu Lys Thr His Gly Glu Val 305 310 315 320 Glu Arg Arg Ile Val Ser Gln Leu Leu Thr Leu Met Asp Gly Leu Lys 325 330 335 Gln Arg Ala His Val Ile Val Met Ala Ala Thr Asn Arg Pro Asn Ser 340 345 350 Ile Asp Pro Ala Leu Arg Arg Phe Gly Arg Phe Asp Arg Glu Val Asp 355 360 365 Ile Gly Ile Pro Asp Ala Thr Gly Arg Leu Glu Ile Leu Gln Ile His 370 375 380 Thr Lys Asn Met Lys Leu Ala Asp Asp Val Asp Leu Glu Gln Val Ala 385 390 395 400 Asn Glu Thr His Gly His Val Gly Ala Asp Leu Ala Ala Leu Cys Ser 405 410 415 Glu Ala Ala Leu Gln Ala Ile Arg Lys Lys Met Asp Leu Ile Asp Leu 420 425 430 Glu Asp Glu Thr Ile Asp Ala Glu Val Met Asn Ser Leu Ala Val Thr 435 440 445 Met Asp Asp Phe Arg Trp Ala Leu Ser Gln Ser Asn Pro Ser Ala Leu 450 455 460 Arg Glu Thr Val Val Glu Val Pro Gln Val Thr Trp Glu Asp Ile Gly 465 470 475 480 Gly Leu Glu Asp Val Lys Arg Glu Leu Gln Glu Leu Val Gln Tyr Pro 485 490 495 Val Glu His Pro Asp Lys Phe Leu Lys Phe Gly Met Thr Pro Ser Lys 500 505 510 Gly Val Leu Phe Tyr Gly Pro Pro Gly Cys Gly Lys Thr Leu Leu Ala 515 520 525 Lys Ala Ile Ala Asn Glu Cys Gln Ala Asn Phe Ile Ser Ile Lys Gly 530 535 540 Pro Glu Leu Leu Thr Met Trp Phe Gly Glu Ser Glu Ala Asn Val Arg 545 550 555 560 Glu Ile Phe Asp Lys Ala Arg Gln Ala Ala Pro Cys Val Leu Phe Phe 565 570 575 Asp Glu Leu Asp Ser Ile Ala Lys Ala Arg Gly Gly Asn Ile Gly Asp 580 585 590 Gly Gly Gly Ala Ala Asp Arg Val Ile Asn Gln Ile Leu Thr Glu Met 595 600 605 Asp Gly Met Ser Thr Lys Lys Asn Val Phe Ile Ile Gly Ala Thr Asn 610 615 620 Arg Pro Asp Ile Ile Asp Pro Ala Ile Leu Arg Pro Gly Arg Leu Asp 625 630 635 640 Gln Leu Ile Tyr Ile Pro Leu Pro Asp Glu Lys Ser Arg Val Ala Ile 645 650 655 Leu Lys Ala Asn Leu Arg Lys Ser Pro Val Ala Lys Asp Val Asp Leu 660 665 670 Glu Phe Leu Ala Lys Met Thr Asn Gly Phe Ser Gly Ala Asp Leu Thr 675 680 685 Glu Ile Cys Gln Arg Ala Cys Lys Leu Ala Ile Arg Glu Ser Ile Glu 690 695 700 Ser Glu Ile Arg Arg Glu Arg Glu Arg Gln Thr Asn Pro Ser Ala Met 705 710 715 720 Glu Val Glu Glu Asp Asp Pro Val Pro Glu Ile Arg Arg Asp His Phe 725 730 735 Glu Glu Ala Met Arg Phe Ala Arg Arg Ser Val Ser Asp Asn Asp Ile 740 745 750 Arg Lys Tyr Glu Met Phe Ala Gln Thr Leu Gln Gln Ser Arg Gly Phe 755 760 765 Gly Ser Phe Arg Phe Pro Ser Gly Asn Gln Gly Gly Ala Gly Pro Ser 770 775 780 Gln Gly Ser Gly Gly Gly Thr Gly Gly Ser Val Tyr Thr Glu Asp Asn 785 790 795 800 Asp Asp Asp Leu Tyr Gly 805 14 142 PRT human 14 Met Gly Ala Pro Thr Leu Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp 1 5 10 15 His Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Cys Ala 20 25 30 Cys Thr Pro Glu Arg Met Ala Glu Ala Gly Phe Ile His Cys Pro Thr 35 40 45 Glu Asn Glu Pro Asp Leu Ala Gln Cys Phe Phe Cys Phe Lys Glu Leu 50 55 60 Glu Gly Trp Glu Pro Asp Asp Asp Pro Ile Glu Glu His Lys Lys His 65 70 75 80 Ser Ser Gly Cys Ala Phe Leu Ser Val Lys Lys Gln Phe Glu Glu Leu 85 90 95 Thr Leu Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Lys Asn Lys 100 105 110 Ile Ala Lys Glu Thr Asn Asn Lys Lys Lys Glu Phe Glu Glu Thr Ala 115 120 125 Lys Lys Val Arg Arg Ala Ile Glu Gln Leu Ala Ala Met Asp 130 135 140 15 1662 DNA human 15 ggcacgaggg cgggacccgt tggcagaggt ggcggcggcg gcatgggtgc cccgacgttg 60 ccccctgcct ggcagccctt tctcaaggac caccgcatct ctacattcaa gaactggccc 120 ttcttggagg gctgcgcctg caccccggag cggatggccg aggctggctt catccactgc 180 cccactgaga acgagccaga cttggcccag tgtttcttct gcttcaagga gctggaaggc 240 tgggagccag atgacgaccc catagaggaa cataaaaagc attcgtccgg ttgcgctttc 300 ctttctgtca agaagcagtt tgaagaatta acccttggtg aatttttgaa actggacaga 360 gaaagagcca agaacaaaat tgcaaaggaa accaacaata agaagaaaga atttgaggaa 420 actgcgaaga aagtgcgccg tgccatcgag cagctggctg ccatggattg aggcctctgg 480 ccggagctgc ctggtcccag agtggctgca ccacttccag ggtttattcc ctggtgccac 540 cagccttcct gtgggcccct tagcaatgtc ttaggaaagg agatcaacat tttcaaatta 600 gatgtttcaa ctgtgctcct gttttgtctt gaaagtggca ccagaggtgc ttctgcctgt 660 gcagcgggtg ctgctggtaa cagtggctgc ttctctctct ctctctcttt tttgggggct 720 catttttgct gttttgattc ccgggcttac caggtgagaa gtgagggagg aagaaggcag 780 tgtccctttt gctagagctg acagctttgt tcgcgtgggc agagccttcc acagtgaatg 840 tgtctggacc tcatgttgtt gaggctgtca cagtcctgag tgtggacttg gcaggtgcct 900 gttgaatctg agctgcaggt tccttatctg tcacacctgt gcctcctcag aggacagttt 960 ttttgttgtt gtgttttttt gttttttttt ttttggtaga tgcatgactt gtgtgtgatg 1020 agagaatgga gacagagtcc ctggctcctc tactgtttaa caacatggct ttcttatttt 1080 gtttgaattg ttaattcaca gaatagcaca aactacaatt aaaactaagc acaaagccat 1140 tctaagtcat tggggaaacg gggtgaactt caggtggatg aggagacaga atagagtgat 1200 aggaagcgtc tggcagatac tccttttgcc actgctgtgt gattagacag gcccagtgag 1260 ccgcggggca catgctggcc gctcctccct cagaaaaagg cagtggccta aatccttttt 1320 aaatgacttg gctcgatgct gtgggggact ggctgggctg ctgcaggccg tgtgtctgtc 1380 agcccaacct tcacatctgt cacgttctcc acacggggga gagacgcagt ccgcccaggt 1440 ccccgctttc tttggaggca gcagctcccg cagggctgaa gtctggcgta agatgatgga 1500 tttgattcgc cctcctccct gtcatagagc tgcagggtgg attgttacag cttcgctgga 1560 aacctctgga ggtcatctcg gctgttcctg agaaataaaa agcctgtcat ttcaaaaaaa 1620 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 1662 16 1248 PRT human 16 Met Asp Ala Lys Ala Arg Asn Cys Leu Leu Gln His Arg Glu Ala Leu 1 5 10 15 Glu Lys Asp Ile Lys Thr Ser Tyr Ile Met Asp His Met Ile Ser Asp 20 25 30 Gly Phe Leu Thr Ile Ser Glu Glu Glu Lys Val Arg Asn Glu Pro Thr 35 40 45 Gln Gln Gln Arg Ala Ala Met Leu Ile Lys Met Ile Leu Lys Lys Asp 50 55 60 Asn Asp Ser Tyr Val Ser Phe Tyr Asn Ala Leu Leu His Glu Gly Tyr 65 70 75 80 Lys Asp Leu Ala Ala Leu Leu His Asp Gly Ile Pro Val Val Ser Ser 85 90 95 Ser Ser Gly Lys Asp Ser Val Ser Gly Ile Thr Ser Tyr Val Arg Thr 100 105 110 Val Leu Cys Glu Gly Gly Val Pro Gln Arg Pro Val Val Phe Val Thr 115 120 125 Arg Lys Lys Leu Val Asn Ala Ile Gln Gln Lys Leu Ser Lys Leu Lys 130 135 140 Gly Glu Pro Gly Trp Val Thr Ile His Gly Met Ala Gly Cys Gly Lys 145 150 155 160 Ser Val Leu Ala Ala Glu Ala Val Arg Asp His Ser Leu Leu Glu Gly 165 170 175 Cys Phe Pro Gly Gly Val His Trp Val Ser Val Gly Lys Gln Asp Lys 180 185 190 Ser Gly Leu Leu Met Lys Leu Gln Asn Leu Cys Thr Arg Leu Asp Gln 195 200 205 Asp Glu Ser Phe Ser Gln Arg Leu Pro Leu Asn Ile Glu Glu Ala Lys 210 215 220 Asp Arg Leu Arg Ile Leu Met Leu Arg Lys His Pro Arg Ser Leu Leu 225 230 235 240 Ile Leu Asp Asp Val Trp Asp Ser Trp Val Leu Lys Ala Phe Asp Ser 245 250 255 Gln Cys Gln Ile Leu Leu Thr Thr Arg Asp Lys Ser Val Thr Asp Ser 260 265 270 Val Met Gly Pro Lys Tyr Val Val Pro Val Glu Ser Ser Leu Gly Lys 275 280 285 Glu Lys Gly Leu Glu Ile Leu Ser Leu Phe Val Asn Met Lys Lys Ala 290 295 300 Asp Leu Pro Glu Gln Ala His Ser Ile Ile Lys Glu Cys Lys Gly Ser 305 310 315 320 Pro Leu Val Val Ser Leu Ile Gly Ala Leu Leu Arg Asp Phe Pro Asn 325 330 335 Arg Trp Glu Tyr Tyr Leu Lys Gln Leu Gln Asn Lys Gln Phe Lys Arg 340 345 350 Ile Arg Lys Ser Ser Ser Tyr Asp Tyr Glu Ala Leu Asp Glu Ala Met 355 360 365 Ser Ile Ser Val Glu Met Leu Arg Glu Asp Ile Lys Asp Tyr Tyr Thr 370 375 380 Asp Leu Ser Ile Leu Gln Lys Asp Val Lys Val Pro Thr Lys Val Leu 385 390 395 400 Cys Ile Leu Trp Asp Met Glu Thr Glu Glu Val Glu Asp Ile Leu Gln 405 410 415 Glu Phe Val Asn Lys Ser Leu Leu Phe Cys Asp Arg Asn Gly Lys Ser 420 425 430 Phe Arg Tyr Tyr Leu His Asp Leu Gln Val Asp Phe Leu Thr Glu Lys 435 440 445 Asn Cys Ser Gln Leu Gln Asp Leu His Lys Lys Ile Ile Thr Gln Phe 450 455 460 Gln Arg Tyr His Gln Pro His Thr Leu Ser Pro Asp Gln Glu Asp Cys 465 470 475 480 Met Tyr Trp Tyr Asn Phe Leu Ala Tyr His Met Ala Ser Ala Lys Met 485 490 495 His Lys Glu Leu Cys Ala Leu Met Phe Ser Leu Asp Trp Ile Lys Ala 500 505 510 Lys Thr Glu Leu Val Gly Pro Ala His Leu Ile His Glu Phe Val Glu 515 520 525 Tyr Arg His Ile Leu Asp Glu Lys Asp Cys Ala Val Ser Glu Asn Phe 530 535 540 Gln Glu Phe Leu Ser Leu Asn Gly His Leu Leu Gly Arg Gln Pro Phe 545 550 555 560 Pro Asn Ile Val Gln Leu Gly Leu Cys Glu Pro Glu Thr Ser Glu Val 565 570 575 Tyr Gln Gln Ala Lys Leu Gln Ala Lys Gln Glu Val Asp Asn Gly Met 580 585 590 Leu Tyr Leu Glu Trp Ile Asn Lys Lys Asn Ile Thr Asn Leu Ser Arg 595 600 605 Leu Val Val Arg Pro His Thr Asp Ala Val Tyr His Ala Cys Phe Ser 610 615 620 Glu Asp Gly Gln Arg Ile Ala Ser Cys Gly Ala Asp Lys Thr Leu Gln 625 630 635 640 Val Phe Lys Ala Glu Thr Gly Glu Lys Leu Leu Glu Ile Lys Ala His 645 650 655 Glu Asp Glu Val Leu Cys Cys Ala Phe Ser Thr Asp Asp Arg Phe Ile 660 665 670 Ala Thr Cys Ser Val Asp Lys Lys Val Lys Ile Trp Asn Ser Met Thr 675 680 685 Gly Glu Leu Val His Thr Tyr Asp Glu His Ser Glu Gln Val Asn Cys 690 695 700 Cys His Phe Thr Asn Ser Ser His His Leu Leu Leu Ala Thr Gly Ser 705 710 715 720 Ser Asp Cys Phe Leu Lys Leu Trp Asp Leu Asn Gln Lys Glu Cys Arg 725 730 735 Asn Thr Met Phe Gly His Thr Asn Ser Val Asn His Cys Arg Phe Ser 740 745 750 Pro Asp Asp Lys Leu Leu Ala Ser Cys Ser Ala Asp Gly Thr Leu Lys 755 760 765 Leu Trp Asp Ala Thr Ser Ala Asn Glu Arg Lys Ser Ile Asn Val Lys 770 775 780 Gln Phe Phe Leu Asn Leu Glu Asp Pro Gln Glu Asp Met Glu Val Ile 785 790 795 800 Val Lys Cys Cys Ser Trp Ser Ala Asp Gly Ala Arg Ile Met Val Ala 805 810 815 Ala Lys Asn Lys Ile Phe Leu Phe Asp Ile His Thr Ser Gly Leu Leu 820 825 830 Gly Glu Ile His Thr Gly His His Ser Thr Ile Gln Tyr Cys Asp Phe 835 840 845 Ser Pro Gln Asn His Leu Ala Val Val Ala Leu Ser Gln Tyr Cys Val 850 855 860 Glu Leu Trp Asn Thr Asp Ser Arg Ser Lys Val Ala Asp Cys Arg Gly 865 870 875 880 His Leu Ser Trp Val His Gly Val Met Phe Ser Pro Asp Gly Ser Ser 885 890 895 Phe Leu Thr Ser Ser Asp Asp Gln Thr Ile Arg Leu Trp Glu Thr Lys 900 905 910 Lys Val Cys Lys Asn Ser Ala Val Met Leu Lys Gln Glu Val Asp Val 915 920 925 Val Phe Gln Glu Asn Glu Val Met Val Leu Ala Val Asp His Ile Arg 930 935 940 Arg Leu Gln Leu Ile Asn Gly Arg Thr Gly Gln Ile Asp Tyr Leu Thr 945 950 955 960 Glu Ala Gln Val Ser Cys Cys Cys Leu Ser Pro His Leu Gln Tyr Ile 965 970 975 Ala Phe Gly Asp Glu Asn Gly Ala Ile Glu Ile Leu Glu Leu Val Asn 980 985 990 Asn Arg Ile Phe Gln Ser Arg Phe Gln His Lys Lys Thr Val Trp His 995 1000 1005 Ile Gln Phe Thr Ala Asp Glu Lys Thr Leu Ile Ser Ser Ser Asp 1010 1015 1020 Asp Ala Glu Ile Gln Val Trp Asn Trp Gln Leu Asp Lys Cys Ile 1025 1030 1035 Phe Leu Arg Gly His Gln Glu Thr Val Lys Asp Phe Arg Leu Leu 1040 1045 1050 Lys Asn Ser Arg Leu Leu Ser Trp Ser Phe Asp Gly Thr Val Lys 1055 1060 1065 Val Trp Asn Ile Ile Thr Gly Asn Lys Glu Lys Asp Phe Val Cys 1070 1075 1080 His Gln Gly Thr Val Leu Ser Cys Asp Ile Ser His Asp Ala Thr 1085 1090 1095 Lys Phe Ser Ser Thr Ser Ala Asp Lys Thr Ala Lys Ile Trp Ser 1100 1105 1110 Phe Asp Leu Leu Leu Pro Leu His Glu Leu Arg Gly His Asn Gly 1115 1120 1125 Cys Val Arg Cys Ser Ala Phe Ser Val Asp Ser Thr Leu Leu Ala 1130 1135 1140 Thr Gly Asp Asp Asn Gly Glu Ile Arg Ile Trp Asn Val Ser Asn 1145 1150 1155 Gly Glu Leu Leu His Leu Cys Ala Pro Leu Ser Glu Glu Gly Ala 1160 1165 1170 Ala Thr His Gly Gly Trp Val Thr Asp Leu Cys Phe Ser Pro Asp 1175 1180 1185 Gly Lys Met Leu Ile Ser Ala Gly Gly Tyr Ile Lys Trp Trp Asn 1190 1195 1200 Val Val Thr Gly Glu Ser Ser Gln Thr Phe Tyr Thr Asn Gly Thr 1205 1210 1215 Asn Leu Lys Lys Ile His Val Ser Pro Asp Phe Lys Thr Tyr Val 1220 1225 1230 Thr Val Asp Asn Leu Gly Ile Leu Tyr Ile Leu Gln Thr Leu Glu 1235 1240 1245 17 3747 DNA human 17 atggatgcaa aagctcgaaa ttgtttgctt caacatagag aagctctgga aaaggacatc 60 aagacatcct acatcatgga tcacatgatt agtgatggat ttttaacaat atcagaagag 120 gaaaaagtaa gaaatgagcc cactcaacag caaagagcag ctatgctgat taaaatgata 180 cttaaaaaag ataatgattc ctacgtatca ttctacaatg ctctactaca tgaaggatat 240 aaagatcttg ctgcccttct ccatgatggc attcctgttg tctcttcttc cagtggtaaa 300 gattcagtta gtggaataac ttcgtatgta aggacagtcc tgtgtgaagg tggagtacca 360 cagaggccag ttgtttttgt cacaaggaag aagctggtga atgcaattca gcagaagctc 420 tccaaattga aaggtgaacc aggatgggtc accatacatg gaatggcagg ctgtgggaag 480 tctgtattag ctgcagaagc tgttagagat cattcccttt tagaaggttg tttcccaggg 540 ggagtgcatt gggtttcagt tgggaaacaa gacaaatctg ggcttctgat gaaactgcag 600 aatctttgca cacggttgga tcaggatgag agtttttccc agaggcttcc acttaatatt 660 gaagaggcta aagaccgtct ccgcattctg atgcttcgca aacacccaag gtctctcttg 720 atcttggatg atgtttggga ctcttgggtg ttgaaagctt ttgacagtca gtgtcagatt 780 cttcttacaa ccagagacaa gagtgttaca gattcagtaa tgggtcctaa atatgtagtc 840 cctgtggaga gttccttagg aaaggaaaaa ggacttgaaa ttttatccct ttttgttaat 900 atgaagaagg cagatttgcc agaacaagct catagtatta taaaagaatg taaaggctct 960 ccccttgtag tatctttaat tggtgcactt ttacgtgatt ttcccaatcg ctgggagtac 1020 tacctcaaac agcttcagaa taagcagttt aagagaataa ggaaatcttc gtcttatgat 1080 tatgaggctc tagatgaagc catgtctata agtgttgaaa tgctcagaga agacatcaaa 1140 gattattaca cagatctttc catccttcag aaggacgtta aggtgcctac aaaggtgtta 1200 tgtattctct gggacatgga aactgaagaa gttgaagaca tactgcagga gtttgtaaat 1260 aagtctcttt tattctgtga tcggaatgga aagtcgtttc gttattattt acatgatctt 1320 caagtagatt ttcttacaga gaagaattgc agccagcttc aggatctaca taagaagata 1380 atcactcagt ttcagagata tcaccagccg catactcttt caccagatca ggaagactgt 1440 atgtattggt acaactttct ggcctatcac atggccagtg ccaagatgca caaggaactt 1500 tgtgctttaa tgttttccct ggattggatt aaagcaaaaa cagaacttgt aggccctgct 1560 catctgattc atgaatttgt ggaatacaga catatactag atgaaaagga ttgtgcagtc 1620 agtgagaatt ttcaggagtt tttatcttta aatggacacc ttcttggacg acagccattt 1680 cctaatattg tacaactggg tctctgtgag ccggaaactt cagaagttta tcagcaagct 1740 aagctgcagg ccaagcagga ggtcgataat ggaatgcttt acctggaatg gataaacaaa 1800 aaaaacatca cgaatctttc ccgcttagtt gtccgccccc acacagatgc tgtttaccat 1860 gcctgctttt ctgaggatgg tcagagaata gcttcttgtg gagctgataa aaccttacag 1920 gtgttcaaag ctgaaacagg agagaaactt ctagaaatca aggctcatga ggatgaagtg 1980 ctttgttgtg cattctctac agatgacaga tttatagcaa cctgctcagt ggataaaaaa 2040 gtgaagattt ggaattctat gactggggaa ctagtacaca cctatgatga gcactcagag 2100 caagtcaatt gctgccattt caccaacagt agtcatcatc ttctcttagc cactgggtca 2160 agtgactgct tcctcaaact ttgggatttg aatcaaaaag aatgtcgaaa taccatgttt 2220 ggtcatacaa attcagtcaa tcactgcaga ttttcaccag atgataagct tttggctagt 2280 tgttcagctg atggaacctt aaagctttgg gatgcgacat cagcaaatga gaggaaaagc 2340 attaatgtga aacagttctt cctaaatttg gaggaccctc aagaggatat ggaagtgata 2400 gtgaagtgtt gttcgtggtc tgctgatggt gcaaggataa tggtggcagc aaaaaataaa 2460 atctttcttt ttgacattca tactagtggc ctattgggag aaatccacac gggccatcac 2520 agcaccatcc agtactgtga cttctcccca caaaaccatt tggcagtggt tgctttgtcc 2580 cagtactgtg tagagttgtg gaatacagac tcacgttcaa aggtggctga ttgcagagga 2640 catttaagtt gggttcatgg tgtgatgttt tctcctgatg gatcatcatt tttgacatct 2700 tctgatgacc agacaatcag gctctgggag acaaagaaag tatgtaagaa ctctgctgta 2760 atgttaaagc aagaagtaga tgttgtgttt caagaaaatg aagtgatggt ccttgcagtt 2820 gaccatataa gacgtctgca actcattaat ggaagaacag gtcagattga ttatctgact 2880 gaagctcaag ttagctgctg ttgcttaagt ccacatcttc agtacattgc atttggagat 2940 gaaaatggag ccattgagat tttagaactt gtaaacaata gaatcttcca gtccaggttt 3000 cagcacaaga aaactgtatg gcacatccag ttcacagccg atgagaagac tcttatttca 3060 agttctgatg atgctgaaat tcaggtatgg aattggcaat tggacaaatg tatctttcta 3120 cgaggccatc aggaaacagt gaaagacttt agactcttga aaaattcaag actgctttct 3180 tggtcatttg atggaacagt gaaggtatgg aatattatta ctggaaataa agaaaaagac 3240 tttgtctgtc accagggtac agtactttct tgtgacattt ctcacgatgc taccaagttt 3300 tcatctacct ctgctgacaa gactgcaaag atctggagtt ttgatctcct tttgccactt 3360 catgaattga ggggccacaa cggctgtgtg cgctgctctg ccttctctgt ggacagtacc 3420 ctgctggcaa cgggagatga caatggagaa atcaggatat ggaatgtctc aaacggtgag 3480 cttcttcatt tgtgtgctcc gctttcagaa gaaggagctg ctacccatgg aggctgggtg 3540 actgaccttt gcttttctcc agatggcaaa atgcttatct ctgctggagg atatattaag 3600 tggtggaacg ttgtcactgg ggaatcctca cagaccttct acacaaatgg aaccaatctt 3660 aagaaaatac acgtgtcccc tgacttcaaa acatatgtga ctgtggataa tcttggtatt 3720 ttatatattt tacagacttt agaataa 3747 18 199 PRT human 18 Met Glu Ala Arg Asp Lys Gln Val Leu Arg Ser Leu Arg Leu Glu Leu 1 5 10 15 Gly Ala Glu Val Leu Val Glu Gly Leu Val Leu Gln Tyr Leu Tyr Gln 20 25 30 Glu Gly Ile Leu Thr Glu Asn His Ile Gln Glu Ile Lys Ala Gln Thr 35 40 45 Thr Gly Leu Arg Lys Thr Met Leu Leu Leu Asp Ile Leu Pro Ser Arg 50 55 60 Gly Pro Lys Ala Phe Asp Thr Phe Leu Asp Ser Leu Gln Glu Phe Pro 65 70 75 80 Trp Val Arg Glu Lys Leu Glu Lys Ala Arg Glu Glu Val Thr Ala Glu 85 90 95 Leu Pro Thr Gly Asp Trp Met Ala Gly Ile Pro Ser His Ile Leu Ser 100 105 110 Ser Ser Pro Ser Asp Gln Gln Ile Asn Gln Leu Ala Gln Lys Leu Gly 115 120 125 Pro Glu Trp Glu Pro Val Val Leu Ser Leu Gly Leu Ser Gln Thr Asp 130 135 140 Ile Tyr Arg Cys Lys Ala Asn His Pro His Asn Val His Ser Gln Val 145 150 155 160 Val Glu Ala Phe Val Arg Trp Arg Gln Arg Phe Gly Lys Gln Ala Thr 165 170 175 Phe Leu Ser Leu His Lys Gly Leu Gln Ala Val Glu Ala Asp Pro Ser 180 185 190 Leu Leu Gln His Met Leu Glu 195 19 600 DNA human 19 atggaagcca gagacaagca ggtactccgc tccctgcgtc tggagctggg tgccgaggta 60 ctggtggaag gactggttct tcagtacctt taccaggaag gaattttgac agaaaaccac 120 attcaagaaa tcaaagctca aaccacaggc ctccggaaga caatgctgtt gctggacatc 180 ctgccttcca ggggccccaa agcttttgac accttcctcg attccctcca ggaatttccc 240 tgggtaagag agaagctgga gaaggcgaga gaggaagtca cagccgagct gcctacaggt 300 gactggatgg ccggaatccc ctcacacatc ctcagcagct cgccatcaga ccagcagatt 360 aaccagctgg ctcagaagct aggcccggag tgggagcccg tggtcctgtc tctgggactg 420 tcccagaccg acatctaccg ctgcaaggcc aaccatcccc acaacgtgca ttcgcaggtg 480 gtggaggcct ttgtccgctg gcgccagcgt tttgggaagc aggccacctt cctaagctta 540 cacaagggcc tccaggcagt ggaggctgat ccctccctgc tccagcacat gctggagtga 600 20 239 PRT human 20 Met Ala Ala Leu Lys Ser Trp Leu Ser Arg Ser Leu Thr Ser Thr Ser 1 5 10 15 Arg Tyr Arg Gln Cys Leu Cys Val Pro Val Val Ala Asn Phe Lys Lys 20 25 30 Arg Cys Phe Ser Glu Leu Ile Arg Pro Trp His Lys Thr Val Thr Ile 35 40 45 Gly Phe Gly Val Thr Leu Cys Ala Val Pro Ile Ala Gln Lys Ser Glu 50 55 60 Pro His Ser Leu Ser Ser Glu Ala Leu Met Arg Arg Ala Val Ser Leu 65 70 75 80 Val Thr Asp Ser Thr Ser Thr Phe Leu Ser Gln Thr Thr Tyr Ala Leu 85 90 95 Ile Glu Ala Ile Thr Glu Tyr Thr Lys Ala Val Tyr Thr Leu Thr Ser 100 105 110 Leu Tyr Arg Gln Tyr Thr Ser Leu Leu Gly Lys Met Asn Ser Glu Glu 115 120 125 Glu Asp Glu Val Trp Gln Val Ile Ile Gly Ala Arg Ala Glu Met Thr 130 135 140 Ser Lys His Gln Glu Tyr Leu Lys Leu Glu Thr Thr Trp Met Thr Ala 145 150 155 160 Val Gly Leu Ser Glu Met Ala Ala Glu Ala Ala Tyr Gln Thr Gly Ala 165 170 175 Asp Gln Ala Ser Ile Thr Ala Arg Asn His Ile Gln Leu Val Lys Leu 180 185 190 Gln Val Glu Glu Val His Gln Leu Ser Arg Lys Ala Glu Thr Lys Leu 195 200 205 Ala Glu Ala Gln Ile Glu Glu Leu Arg Gln Lys Thr Gln Glu Glu Gly 210 215 220 Glu Glu Arg Ala Glu Ser Glu Gln Glu Ala Tyr Leu Arg Glu Asp 225 230 235 21 1351 DNA human 21 ggcgtccgcg cgctgcacaa tggcggctct gaagagttgg ctgtcgcgca gcctgacttc 60 tacttccagg tacagacagt gtttgtgtgt tcctgttgtg gctaacttta agaagcggtg 120 tttctcagaa ttgataagac catggcacaa aactgtgacg attggctttg gagtaaccct 180 gtgtgcggtt cctattgcac agaaatcaga gcctcattcc cttagtagtg aagcattgat 240 gaggagagca gtgtctttgg taacagatag cacctctacc tttctctctc agaccacata 300 tgcgttgatt gaagctatta ctgaatatac taaggctgtt tataccttaa cttctcttta 360 ccgacaatat acaagtttac ttgggaaaat gaattcagag gaggaagatg aagtgtggca 420 ggtgatcata ggagccagag ctgagatgac ttcaaaacac caagagtact tgaagctgga 480 aaccacttgg atgactgcag ttggtctttc agagatggca gcagaagctg catatcaaac 540 tggcgcagat caggcctcta taaccgccag gaatcacatt cagctggtga aactgcaggt 600 ggaagaggtg caccagctct cccggaaagc agaaaccaag ctggcagaag cacagataga 660 agagctccgt cagaaaacac aggaggaagg ggaggagcgg gctgagtcgg agcaggaggc 720 ctacctgcgt gaggattgag ggcctgagca cactgccctg tctccccact cagtggggaa 780 agcaggggca gatgccaccc tgcccagggt tggcatgact gtctgtgcac cgagaagagg 840 cggcagatcc tgccctggcc aatcaggcga gacgcctttg tgagctgtga gtgcctcctg 900 tggtctcagg cttgcgctgg acctggttct tagcccttgg gcactgcacc ctgtttaaca 960 tttcacccca ctctgtacag ctgctcttac ccattttttt tacctcacac ccaaagcatt 1020 ttgcctacct gggtcagaga gaggagtcct ttttgtcatg cccttaagtt cagcaactgt 1080 ttaacctgtt ttcagtctta tttacgtcgt caaaaatgat ttagtacttg ttccctctgt 1140 tgggatgcca gttgtggcag ggggagggga acctgtccag tttgtacgat ttctttgtat 1200 gtatttctga tgtgttctct gatctgcccc cactgtcctg tgaggacagc tgaggccaag 1260 gagtgaaaaa cctattacta ctaagagaag gggtgcagag tgtttacctg gtgctctcaa 1320 caggacttaa catcaacagg acttaacaca g 1351 22 1403 PRT human 22 Met Ala Thr Gln Gln Lys Ala Ser Asp Glu Arg Ile Ser Gln Phe Asp 1 5 10 15 His Asn Leu Leu Pro Glu Leu Ser Ala Leu Leu Gly Leu Asp Ala Val 20 25 30 Gln Leu Ala Lys Glu Leu Glu Glu Glu Glu Gln Lys Glu Arg Ala Lys 35 40 45 Met Gln Lys Gly Tyr Asn Ser Gln Met Arg Ser Glu Ala Lys Arg Leu 50 55 60 Lys Thr Phe Val Thr Tyr Glu Pro Tyr Ser Ser Trp Ile Pro Gln Glu 65 70 75 80 Met Ala Ala Ala Gly Phe Tyr Phe Thr Gly Val Lys Ser Gly Ile Gln 85 90 95 Cys Phe Cys Cys Ser Leu Ile Leu Phe Gly Ala Gly Leu Thr Arg Leu 100 105 110 Pro Ile Glu Asp His Lys Arg Phe His Pro Asp Cys Gly Phe Leu Leu 115 120 125 Asn Lys Asp Val Gly Asn Ile Ala Lys Tyr Asp Ile Arg Val Lys Asn 130 135 140 Leu Lys Ser Arg Leu Arg Gly Gly Lys Met Arg Tyr Gln Glu Glu Glu 145 150 155 160 Ala Arg Leu Ala Ser Phe Arg Asn Trp Pro Phe Tyr Val Gln Gly Ile 165 170 175 Ser Pro Cys Val Leu Ser Glu Ala Gly Phe Val Phe Thr Gly Lys Gln 180 185 190 Asp Thr Val Gln Cys Phe Ser Cys Gly Gly Cys Leu Gly Asn Trp Glu 195 200 205 Glu Gly Asp Asp Pro Trp Lys Glu His Ala Lys Trp Phe Pro Lys Cys 210 215 220 Glu Phe Leu Arg Ser Lys Lys Ser Ser Glu Glu Ile Thr Gln Tyr Ile 225 230 235 240 Gln Ser Tyr Lys Gly Phe Val Asp Ile Thr Gly Glu His Phe Val Asn 245 250 255 Ser Trp Val Gln Arg Glu Leu Pro Met Ala Ser Ala Tyr Cys Asn Asp 260 265 270 Ser Ile Phe Ala Tyr Glu Glu Leu Arg Leu Asp Ser Phe Lys Asp Trp 275 280 285 Pro Arg Glu Ser Ala Val Gly Val Ala Ala Leu Ala Lys Ala Gly Leu 290 295 300 Phe Tyr Thr Gly Ile Lys Asp Ile Val Gln Cys Phe Ser Cys Gly Gly 305 310 315 320 Cys Leu Glu Lys Trp Gln Glu Gly Asp Asp Pro Leu Asp Asp His Thr 325 330 335 Arg Cys Phe Pro Asn Cys Pro Phe Leu Gln Asn Met Lys Ser Ser Ala 340 345 350 Glu Val Thr Pro Asp Leu Gln Ser Arg Gly Glu Leu Cys Glu Leu Leu 355 360 365 Glu Thr Thr Ser Glu Ser Asn Leu Glu Asp Ser Ile Ala Val Gly Pro 370 375 380 Ile Val Pro Glu Met Ala Gln Gly Glu Ala Gln Trp Phe Gln Glu Ala 385 390 395 400 Lys Asn Leu Asn Glu Gln Leu Arg Ala Ala Tyr Thr Ser Ala Ser Phe 405 410 415 Arg His Met Ser Leu Leu Asp Ile Ser Ser Asp Leu Ala Thr Asp His 420 425 430 Leu Leu Gly Cys Asp Leu Ser Ile Ala Ser Lys His Ile Ser Lys Pro 435 440 445 Val Gln Glu Pro Leu Val Leu Pro Glu Val Phe Gly Asn Leu Asn Ser 450 455 460 Val Met Cys Val Glu Gly Glu Ala Gly Ser Gly Lys Thr Val Leu Leu 465 470 475 480 Lys Lys Ile Ala Phe Leu Trp Ala Ser Gly Cys Cys Pro Leu Leu Asn 485 490 495 Arg Phe Gln Leu Val Phe Tyr Leu Ser Leu Ser Ser Thr Arg Pro Asp 500 505 510 Glu Gly Leu Ala Ser Ile Ile Cys Asp Gln Leu Leu Glu Lys Glu Gly 515 520 525 Ser Val Thr Glu Met Cys Met Arg Asn Ile Ile Gln Gln Leu Lys Asn 530 535 540 Gln Val Leu Phe Leu Leu Asp Asp Tyr Lys Glu Ile Cys Ser Ile Pro 545 550 555 560 Gln Val Ile Gly Lys Leu Ile Gln Lys Asn His Leu Ser Arg Thr Cys 565 570 575 Leu Leu Ile Ala Val Arg Thr Asn Arg Ala Arg Asp Ile Arg Arg Tyr 580 585 590 Leu Glu Thr Ile Leu Glu Ile Lys Ala Phe Pro Phe Tyr Asn Thr Val 595 600 605 Cys Ile Leu Arg Lys Leu Phe Ser His Asn Met Thr Arg Leu Arg Lys 610 615 620 Phe Met Val Tyr Phe Gly Lys Asn Gln Ser Leu Gln Lys Ile Gln Lys 625 630 635 640 Thr Pro Leu Phe Val Ala Ala Ile Cys Ala His Trp Phe Gln Tyr Pro 645 650 655 Phe Asp Pro Ser Phe Asp Asp Val Ala Val Phe Lys Ser Tyr Met Glu 660 665 670 Arg Leu Ser Leu Arg Asn Lys Ala Thr Ala Glu Ile Leu Lys Ala Thr 675 680 685 Val Ser Ser Cys Gly Glu Leu Ala Leu Lys Gly Phe Phe Ser Cys Cys 690 695 700 Phe Glu Phe Asn Asp Asp Asp Leu Ala Glu Ala Gly Val Asp Glu Asp 705 710 715 720 Glu Asp Leu Thr Met Cys Leu Met Ser Lys Phe Thr Ala Gln Arg Leu 725 730 735 Arg Pro Phe Tyr Arg Phe Leu Ser Pro Ala Phe Gln Glu Phe Leu Ala 740 745 750 Gly Met Arg Leu Ile Glu Leu Leu Asp Ser Asp Arg Gln Glu His Gln 755 760 765 Asp Leu Gly Leu Tyr His Leu Lys Gln Ile Asn Ser Pro Met Met Thr 770 775 780 Val Ser Ala Tyr Asn Asn Phe Leu Asn Tyr Val Ser Ser Leu Pro Ser 785 790 795 800 Thr Lys Ala Gly Pro Lys Ile Val Ser His Leu Leu His Leu Val Asp 805 810 815 Asn Lys Glu Ser Leu Glu Asn Ile Ser Glu Asn Asp Asp Tyr Leu Lys 820 825 830 His Gln Pro Glu Ile Ser Leu Gln Met Gln Leu Leu Arg Gly Leu Trp 835 840 845 Gln Ile Cys Pro Gln Ala Tyr Phe Ser Met Val Ser Glu His Leu Leu 850 855 860 Val Leu Ala Leu Lys Thr Ala Tyr Gln Ser Asn Thr Val Ala Ala Cys 865 870 875 880 Ser Pro Phe Val Leu Gln Phe Leu Gln Gly Arg Thr Leu Thr Leu Gly 885 890 895 Ala Leu Asn Leu Gln Tyr Phe Phe Asp His Pro Glu Ser Leu Ser Leu 900 905 910 Leu Arg Ser Ile His Phe Pro Ile Arg Gly Asn Lys Thr Ser Pro Arg 915 920 925 Ala His Phe Ser Val Leu Glu Thr Cys Phe Asp Lys Ser Gln Val Pro 930 935 940 Thr Ile Asp Gln Asp Tyr Ala Ser Ala Phe Glu Pro Met Asn Glu Trp 945 950 955 960 Glu Arg Asn Leu Ala Glu Lys Glu Asp Asn Val Lys Ser Tyr Met Asp 965 970 975 Met Gln Arg Arg Ala Ser Pro Asp Leu Ser Thr Gly Tyr Trp Lys Leu 980 985 990 Ser Pro Lys Gln Tyr Lys Ile Pro Cys Leu Glu Val Asp Val Asn Asp 995 1000 1005 Ile Asp Val Val Gly Gln Asp Met Leu Glu Ile Leu Met Thr Val 1010 1015 1020 Phe Ser Ala Ser Gln Arg Ile Glu Leu His Leu Asn His Ser Arg 1025 1030 1035 Gly Phe Ile Glu Ser Ile Arg Pro Ala Leu Glu Leu Ser Lys Ala 1040 1045 1050 Ser Val Thr Lys Cys Ser Ile Ser Lys Leu Glu Leu Ser Ala Ala 1055 1060 1065 Glu Gln Glu Leu Leu Leu Thr Leu Pro Ser Leu Glu Ser Leu Glu 1070 1075 1080 Val Ser Gly Thr Ile Gln Ser Gln Asp Gln Ile Phe Pro Asn Leu 1085 1090 1095 Asp Lys Phe Leu Cys Leu Lys Glu Leu Ser Val Asp Leu Glu Gly 1100 1105 1110 Asn Ile Asn Val Phe Ser Val Ile Pro Glu Glu Phe Pro Asn Phe 1115 1120 1125 His His Met Glu Lys Leu Leu Ile Gln Ile Ser Ala Glu Tyr Asp 1130 1135 1140 Pro Ser Lys Leu Val Lys Leu Ile Gln Asn Ser Pro Asn Leu His 1145 1150 1155 Val Phe His Leu Lys Cys Asn Phe Phe Ser Asp Phe Gly Ser Leu 1160 1165 1170 Met Thr Met Leu Val Ser Cys Lys Lys Leu Thr Glu Ile Lys Phe 1175 1180 1185 Ser Asp Ser Phe Phe Gln Ala Val Pro Phe Val Ala Ser Leu Pro 1190 1195 1200 Asn Phe Ile Ser Leu Lys Ile Leu Asn Leu Glu Gly Gln Gln Phe 1205 1210 1215 Pro Asp Glu Glu Thr Ser Glu Lys Phe Ala Tyr Ile Leu Gly Ser 1220 1225 1230 Leu Ser Asn Leu Glu Glu Leu Ile Leu Pro Thr Gly Asp Gly Ile 1235 1240 1245 Tyr Arg Val Ala Lys Leu Ile Ile Gln Gln Cys Gln Gln Leu His 1250 1255 1260 Cys Leu Arg Val Leu Ser Phe Phe Lys Thr Leu Asn Asp Asp Ser 1265 1270 1275 Val Val Glu Ile Ala Lys Val Ala Ile Ser Gly Gly Phe Gln Lys 1280 1285 1290 Leu Glu Asn Leu Lys Leu Ser Ile Asn His Lys Ile Thr Glu Glu 1295 1300 1305 Gly Tyr Arg Asn Phe Phe Gln Ala Leu Asp Asn Met Pro Asn Leu 1310 1315 1320 Gln Glu Leu Asp Ile Ser Arg His Phe Thr Glu Cys Ile Lys Ala 1325 1330 1335 Gln Ala Thr Thr Val Lys Ser Leu Ser Gln Cys Val Leu Arg Leu 1340 1345 1350 Pro Arg Leu Ile Arg Leu Asn Met Leu Ser Trp Leu Leu Asp Ala 1355 1360 1365 Asp Asp Ile Ala Leu Leu Asn Val Met Lys Glu Arg His Pro Gln 1370 1375 1380 Ser Lys Tyr Leu Thr Ile Leu Gln Lys Trp Ile Leu Pro Phe Ser 1385 1390 1395 Pro Ile Ile Gln Lys 1400 

What is claimed is:
 1. A nucleic acid which specifically hybridizes to a nucleic acid encoding an inhibitor-of-apoptosis protein.
 2. The nucleic acid of claim 1, wherein the nucleic acid is complementary to the nucleic acid encoding the inhibitor-of-apoptosis protein.
 3. The nucleic acid of claim 1, wherein the nucleic acid has a length of from about 15 nucleotides to about 25 nucleotides.
 4. The nucleic acid of claim 1, wherein the inhibitor-of-apoptosis protein is selected from the group consisting of MIAP1, MIAP2, MIAP3, CIAP1, CIAP2, and XIAP.
 5. The nucleic acid of claim 1, wherein the nucleic acid specifically hybridizes to the portion of the nucleic acid encoding MIAP3 beginning with the adenosine at position 769 and ending with the guanosine at position
 791. 6. A composition comprising the nucleic acid of claim 1 and a carrier.
 7. The composition of claim 6, wherein the composition comprises nucleic acids which specifically hybridize to nucleic acids encoding a plurality of inhibitor-of-apoptosis proteins.
 8. The composition of claim 7, wherein the inhibitor-of-apoptosis proteins comprise CIAP1, CIAP2 and XIAP.
 9. The composition of claim 7, wherein the inhibitor-of-apoptosis proteins comprise CIAP1 and XIAP.
 10. The composition of claim 7, wherein the inhibitor-of-apoptosis proteins comprise CIAP2 and XIAP.
 11. The composition of claim 7, wherein the inhibitor-of-apoptosis proteins comprise CIAP1 and CIAP2.
 12. The composition of claim 6, wherein the carrier comprises a diluent, an adjuvant, a virus, a liposome, a microencapsule, a neuronal cell receptor ligand, a neuronal-specific virus, a polymer-encapsulated cell or a retroviral vector.
 13. The composition of claim 6, wherein the carrier is an aerosol, an intravenous carrier, an oral carrier or a topical carrier.
 14. A method for inducing a cell's death which comprises contacting the cell with the nucleic acid of claim 1 under conditions permitting the nucleic acid to enter the cell.
 15. The method of claim 14, further comprising contacting the cell with nucleic acids which specifically hybridize to nucleic acids encoding a plurality of inhibitor-of-apoptosis proteins.
 16. The method of claim 15, wherein the inhibitor of apoptosis proteins comprise CIAP1, CIAP2 and XIAP.
 17. The method of claim 15, wherein the inhibitor of apoptosis proteins comprise CIAP1 and XIAP.
 18. The method of claim 15, wherein the inhibitor of apoptosis proteins comprise CIAP2 and XIAP.
 19. The method of claim 15, wherein the inhibitor of apoptosis proteins comprise CIAP1 and CIAP2.
 20. The method of claim 14, wherein the conditions permitting the nucleic acid to enter the cell comprise the use of a vector, a liposome, a mechanical means or an electrical means.
 21. The method of claim 20, wherein the vector is selected from the group consisting of an adenovirus vector, an adeno-associated virus vector, an Epstein-Barr virus vector, a Herpes virus vector, an attenuated HIV vector, a retroviral vector and a vaccinia virus vector.
 22. The method of claim 20, wherein the liposome is an antibody-coated liposome.
 23. A method for treating a subject afflicted with cancer which comprises administering to the subject a therapeutically effective amount of the nucleic acid of claim
 1. 24. The method of claim 23, wherein the cancer is selected from the group consisting of acute lymphocytic leukemia, acute myelogenous leukemia, lung cancer, breast cancer, ovarian cancer, prostate cancer, lymphoma, Hodgkin's disease, malignant melanoma, neuroblastoma, renal cell carcinoma and squamous cell carcinoma.
 25. The method of claim 23, wherein the cancer is a tumor.
 26. The method of claim 23, wherein the subject is a mammal.
 27. The method of claim 26, wherein the subject is a human.
 28. An nucleic acid that specifically hybridizes to a nucleic acid which encodes a protein, other than caspase-2, that induces cell death.
 29. The nucleic acid of claim 28, wherein the nucleic acid is complementary to the nucleic acid encoding the protein that induces cell death.
 30. The nucleic acid of claim 28, wherein the nucleic acid has a length of from about 15 nucleotides to about 25 nucleotides.
 31. The nucleic acid of claim 28, wherein the protein is selected from the group consisting of APAF1, RAIDD, and Diablo/SMAC.
 32. The nucleic acid of claim 28 which specifically hybridizes to a nucleic acid encoding the protein APAF1.
 33. The nucleic acid of claim 28, wherein the nucleic acid specifically hybridizes to the portion of the nucleic acid encoding APAF-1 beginning with the cytosine at position 576 and ending with the adenosine at position
 596. 34. The nucleic acid of claim 28 which specifically hybridizes to a nucleic acid encoding the protein RAIDD.
 35. The nucleic acid of claim 28, wherein the nucleic acid specifically hybridizes to the portion of the nucleic acid encoding RAIDD beginning with the guanosine at position 110 and ending with the adenosine at position
 130. 36. The nucleic acid of claim 28 which specifically hybridizes to a nucleic acid encoding the protein Diablo/SMAC.
 37. The nucleic acid of claim 28, wherein the nucleic acid specifically hybridizes to the portion of the nucleic acid encoding Diablo/SMAC beginning with the thymidine at position 1 and ending with the thymidine at position
 21. 38. A composition comprising the nucleic acid of claim 28 and a carrier.
 39. The composition of claim 38, wherein the composition comprises nucleic acids which specifically hybridize to nucleic acids encoding a plurality of proteins that induce cell death.
 40. The composition of claim 39, wherein the proteins comprise APAF-1 and Diablo/SMAC.
 41. The composition of claim 39, wherein the proteins comprise APAF-1, Diablo/SMAC and caspase-9.
 42. The composition of claim 39, wherein the proteins comprise APAF-1, Diablo/SMAC and caspase-7.
 43. The composition of claim 39, wherein the proteins comprise caspase-2 and RAIDD.
 44. The composition of claim 39, wherein the proteins comprise caspase-8 and RAIDD.
 45. The composition of claim 39, wherein the proteins comprise caspase-8, RAIDD and caspase-3.
 46. The composition of claim 39, wherein the proteins comprise caspase-2 and caspase-9.
 47. The composition of claim 38, wherein the carrier comprises a diluent, an adjuvant, a virus, a liposome, a microencapsule, a neuronal cell receptor ligand, a neuronal-specific virus, a polymer-encapsulated cell or a retroviral vector.
 48. The composition of claim 38, wherein the carrier is an aerosol, an intravenous carrier, an oral carrier or a topical carrier.
 49. A method for inhibiting a cell's death which comprises contacting the cell with the nucleic acid of claim 28 under conditions permitting the nucleic acid to enter the cell.
 50. A method for inhibiting a neuronal cell's death which comprises contacting the cell with the nucleic acid of claim 28 under conditions permitting the nucleic acid to enter the cell.
 51. The method of claim 49 or 50, further comprising contacting the cell with nucleic acids which specifically hybridize to nucleic acids encoding a plurality of proteins that induce cell death.
 52. The method of claim 51, wherein the proteins that induce cell death comprise APAF-1 and Diablo/SMAC.
 53. The method of claim 51, wherein the proteins that induce cell death comprise APAF-1, Diablo/SMAC and caspase-9.
 54. The method of claim 51, wherein the proteins that induce cell death comprise APAF-1, Diablo/SMAC and caspase-7.
 55. The method of claim 51, wherein the proteins that induce cell death comprise caspase-2 and RAIDD.
 56. The method of claim 51, wherein the proteins that induce cell death comprise caspase-8 and RAIDD.
 57. The method of claim 51, wherein the proteins that induce cell death comprise caspase-8, RAIDD and caspase-3.
 58. The method of claim 51, wherein the proteins that induce cell death comprise caspase-2 and caspase-9.
 59. The method of claim 49 or 50, wherein the conditions permitting the nucleic acid to enter the cell comprise the use of a vector, a liposome, a mechanical means or an electrical means.
 60. The method of claim 59, wherein the vector is selected from the group consisting of an adenovirus vector, an adeno-associated virus vector, an Epstein-Barr virus vector, a Herpes virus vector, an attenuated HIV vector, a retroviral vector and a vaccinia virus vector.
 61. The method of claim 59, wherein the liposome is an antibody-coated liposome.
 62. A method for treating a neurodegenerative disorder in a subject which comprises administering to the subject a therapeutically effective amount of the nucleic acid of claim
 28. 63. The method of claim 62, wherein the neurodegenerative disorder is a brain disorder or a central nervous system disorder.
 64. A method for treating a heart disorder in a subject which comprises administering to the subject a therapeutically effective amount of the nucleic acid of claim
 28. 65. The method of claim 64, wherein the heart disorder is cardiomyopathy.
 66. The method of claim 49, 50, 62 or 64, wherein the subject is a mammal.
 67. The method of claim 66, wherein the subject is a human. 